Anti-microRNA oligonucleotide molecules

ABSTRACT

The invention relates to isolated anti-microRNA molecules. In another embodiment, the invention relates to an isolated microRNA molecule. In yet another embodiment, the invention provides a method for inhibiting microRNP activity in a cell.

This application is a divisional of U.S. application Ser. No. 10/589,449filed on Apr. 27, 2007 now U.S. Pat. No. 7,772,389, which is a U.S.National Phase Application of International Application No.PCT/US05/04714 filed on Feb. 11, 2005 and asserts priority to U.S.application Ser. No. 10/845,057 filed on May 13, 2004 now abandoned,which is a continuing application of U.S. application Ser. No.10/778,908 filed on Feb. 13, 2004 now abandoned; all of which are herebyincorporated by reference in their entirety.

The invention claimed herein was made with the help of grant number 1R01 GM068476-01 from NIH/NIGMS. The U.S. government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

RNA silencing is a fundamental mechanism of gene regulation that usesdouble-stranded RNA (dsRNA) derived 21- to 28-nucleotide (nt) small RNAsto guide mRNA degradation, control mRNA translation or chromatinmodification. Recently, several hundred novel genes were identified inplants and animals that encode transcripts that contain short dsRNAhairpins.

Defined 22-nt RNAs, referred to as microRNAs (miRNAs), are reported tobe excised by dsRNA specific endonucleases from the hairpin precursors.The miRNAs are incorporated into ribonucleoprotein particles (miRNPs).

Plant miRNAs target mRNAs containing sequence segments with highcomplementarity for degradation or suppress translation of partiallycomplementary mRNAs. Animal miRNAs appear to act predominantly astranslational repressors. However, animal miRNAs have also been reportedto guide RNA degradation. This indicates that animal miRNPs act likesmall interfering RNA (siRNA)-induced silencing complexes (RISCs).

Understanding the biological function of miRNAs requires knowledge oftheir mRNA targets. Bioinformatic approaches have been used to predictmRNA targets, among which transcription factors and proapoptotic geneswere prominent candidates. Processes such as Notch signaling, cellproliferation, morphogenesis and axon guidance appear to be controlledby miRNA genes.

Therefore, there is a need for materials and methods that can helpelucidate the function of known and future microRNAs. Due to the abilityof microRNAs to induce RNA degradation or repress translation of mRNAwhich encode important proteins, there is also a need for novelcompositions for inhibiting microRNA-induced cleavage or repression ofmRNAs.

SUMMARY THE INVENTION

In one embodiment, the invention provides an isolated single strandedanti-microRNA molecule comprising a minimum of ten moieties and amaximum of fifty moieties on a molecular backbone, the molecularbackbone comprising backbone units, each moiety comprising a base bondedto a backbone unit, each base forming a Watson-Crick base pair with acomplementary base wherein at least ten contiguous bases have the samesequence as a sequence of bases in any one of the anti-microRNAmolecules shown in Tables 1-4, except that up to thirty percent of thebases pairs may be wobble base pairs, and up to 10% of the contiguousbases may be additions, deletions, mismatches, or combinations thereof;no more than fifty percent of the contiguous moieties containdeoxyribonucleotide backbone units; the moiety in the molecule at theposition corresponding to position 11 of the microRNA isnon-complementary; and the molecule is capable of inhibiting microRNPactivity.

In another embodiment, the invention provides a method for inhibitingmicroRNP activity in a cell, the microRNP comprising a microRNAmolecule, the microRNA molecule comprising a sequences of basescomplementary of the sequence of bases in a single strandedanti-microRNA molecule, the method comprising introducing into the cellthe single-stranded anti-microRNA molecule comprising a sequence of aminimum of ten moieties and a maximum of fifty moieties on a molecularbackbone, the molecular backbone comprising backbone units, each moietycomprising a base bonded to a backbone unit, each base forming aWatson-Crick base pair with a complementary base, wherein at least tencontiguous bases of the anti-microRNA molecule are complementary to themicroRNA, except that up to thirty percent of the bases may besubstituted by wobble base pairs, and up to ten percent of the at leastten moieties may be additions, deletions, mismatches, or combinationsthereof; no more than fifty percent of the contiguous moieties containdeoxyribonucleotide backbone units; and the moiety in the molecule atthe position corresponding to position 11 of the microRNA isnon-complementary.

In another embodiment, the invention provides an isolated microRNAmolecule comprising a minimum of ten moieties and a maximum of fiftymoieties on a molecular backbone, the molecular backbone comprisingbackbone units, each moiety comprising a base bonded to a backbone unit,wherein at least ten contiguous bases have the same sequence as asequence of bases in any one of the microRNA molecules shown in Table 2,except that up to thirty percent of the bases pairs may be wobble basepairs, and up to 10% of the contiguous bases may be additions,deletions, mismatches, or combinations thereof; and no more than fiftypercent of the contiguous moieties contain deoxyribonucleotide backboneunits.

In another embodiment, the invention provides an isolated microRNAmolecule comprising a minimum of ten moieties and a maximum of fiftymoieties on a molecular backbone, the molecular backbone comprisingbackbone units, each moiety comprising a base bonded to a backbone unit,wherein at least ten contiguous bases have any one of the microRNAsequences shown in Tables 1, 3 and 4, except that up to thirty percentof the bases pairs may be wobble base pairs, and up to 10% of thecontiguous bases may be additions, deletions, mismatches, orcombinations thereof; no more than fifty percent of the contiguousmoieties contain deoxyribonucleotide backbone units; and is modified forincreased nuclease resistance.

In yet another embodiment, the invention provides an isolated singlestranded anti-microRNA molecule comprising a minimum of ten moieties anda maximum of fifty moieties on a molecular backbone, the molecularbackbone comprising backbone units, each moiety comprising a base bondedto a backbone unit, each base forming a Watson-Crick base pair with acomplementary base wherein at least ten contiguous bases have the samesequence as a sequence of bases in any one of the anti-microRNAmolecules shown in Tables 1-4, except that up to thirty percent of thebases pairs may be wobble base pairs, and up to 10% of the contiguousbases may be additions, deletions, mismatches, or combinations thereof;no more than fifty percent of the contiguous moieties containdeoxyribonucleotide backbone units; and the molecule is capable ofinhibiting microRNP activity.

In yet a further embodiment, the invention provides a method forinhibiting microRNP activity in a cell, the microRNP comprising amicroRNA molecule, the microRNA molecule comprising a sequences of basescomplementary of the sequence of bases in a single strandedanti-microRNA molecule, the method comprising introducing into the cellthe single-stranded anti-microRNA molecule comprising a sequence of aminimum of ten moieties and a maximum of fifty moieties on a molecularbackbone, the molecular backbone comprising backbone units, each moietycomprising a base bonded to a backbone unit, each base forming aWatson-Crick base pair with a complementary base, wherein at least tencontiguous bases of the anti-microRNA molecule are complementary to themicroRNA, except that up to thirty percent of the bases may besubstituted by wobble base pairs, and up to ten percent of the at leastten moieties may be additions, deletions, mismatches, or combinationsthereof; and no more than fifty percent of the contiguous moietiescontain deoxyribonucleotide backbone units.

DESCRIPTION OF THE FIGURES

FIG. 1. shows the modified nucleotide units discussed in thespecification. B denotes any one of the following nucleic acid bases:adenosine, cytidine, guanosine, thymine, or uridine.

FIG. 2. Antisense 2′-O-methyl oligoribonucleotide specifically inhibitmiR-21 guided cleavage activity in HeLa cell S100 cytoplasmic extracts.The black bar to the left of the RNase T1 ladder represents the regionof the target RNA complementary to miR-21. Oligonucleotidescomplementary to miR-21 were pre-incubated in S100 extracts prior to theaddition of ³²P-cap-labelled cleavage substrate. Cleavage bands and T1hydrolysis bands appear as doublets after a 1-nt slipping of the T7 RNApolymerase near the middle of the transcript indicated by the asterisk.

FIG. 3. Antisense 2′-O-methyl oligoribonucleotides interfere withendogenous miR-21 RNP cleavage in HeLa cells. HeLa cells weretransfected with pHcRed and pEGFP or its derivatives, with or withoutinhibitory or control oligonucleotides. EGFP and HcRed proteinfluorescence were excited and recorded individually by fluorescencemicroscopy 24 h after transfection. Co-expression of co-transfectedreporter plasmids was documented by superimposing of the fluorescenceimages in the right panel.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated single stranded anti-microRNAmolecule. The molecule comprises a minimum number of ten moieties,preferably a minimum of thirteen, more preferably a minimum of fifteen,even more preferably a minimum of 18, and most preferably a minimum of21 moieties.

The anti-microRNA molecule comprises a maximum number of fifty moieties,preferably a maximum of forty, more preferably a maximum of thirty, evenmore preferably a maximum of twenty-five, and most preferably a maximumof twenty-three moieties. A suitable range of minimum and maximum numberof moieties may be obtained by combining any of the above minima withany of the above maxima.

Each moiety comprises a base bonded to a backbone unit. In thisspecification, a base refers to any one of the nucleic acid basespresent in DNA or RNA. The base can be a purine or pyrimidine. Examplesof purine bases include adenine (A) and guanine (G). Examples ofpyrimidine bases include thymine (T), cytosine (C) and uracil (U). Eachbase of the moiety forms a Watson-Crick base pair with a complementarybase.

Watson-Crick base pairs as used herein refers to the hydrogen bondinginteraction between, for example, the following bases: adenine andthymine (A=T); adenine and uracil (A=U); and cytosine and guanine (C=G).The adenine can be replaced with 2,6-diaminopurine without compromisingbase-pairing.

The backbone unit may be any molecular unit that is able stably to bindto a base and to form an oligomeric chain. Suitable backbone units arewell known to those in the art.

For example, suitable backbone units include sugar-phosphate groups,such as the sugar-phosphate groups present in ribonucleotides,deoxyribonucleotides, phosphorothioate deoxyribose groups, N′3-N′5phosphoroamidate deoxyribose groups, 2′O-alkyl-ribose phosphate groups,2′-O-alkyl-alkoxy ribose phosphate groups, ribose phosphate groupcontaining a methylene bridge, 2′-Fluororibose phosphate groups,morpholino phosphoroamidate groups, cyclohexene groups, tricyclophosphate groups, and amino acid molecules.

In one embodiment, the anti-microRNA molecule comprises at least onemoiety which is a ribonucleotide moiety or a deoxyribonucleotide moiety.

In another embodiment, the anti-microRNA molecule comprises at least onemoiety which confers increased nuclease resistance. The nuclease can bean exonuclease, an endonuclease, or both. The exonuclease can be a 3′→5′exonuclease or a 5′→3′ exonuclease. Examples of 3′→5′ human exonucleaseinclude PNPT1, Werner syndrome helicase, RRP40, RRP41, RRP42, RRP45, andRRP46. Examples of 5′→3′ exonuclease include XRN2, and FEN1. Examples ofendonucleases include Dicer, Drosha, RNase4, Ribonuclease P,Ribonuclease H1, DHP1, ERCC-1 and OGG1. Examples of nucleases whichfunction as both an exonuclease and an endonuclease include APE1 andEXO1.

An anti-microRNA molecule comprising at least one moiety which confersincreased nuclease resistance means a sequence of moieties wherein atleast one moiety is not recognized by a nuclease. Therefore, thenuclease resistance of the molecule is increased compared to a sequencecontaining only unmodified ribonucleotide, unmodifieddeoxyribonucleotide or both. Such modified moieties are well known inthe art, and were reviewed, for example, by Kurreck, Eur. J. Biochem.270, 1628-1644 (2003).

A modified moiety can occur at any position in the anti-microRNAmolecule. For example, to protect the anti-microRNA molecule against3′→5′ exonucleases, the molecule can have at least one modified moietyat the 3′ end of the molecule and preferably at least two modifiedmoieties at the 3′ end. If it is desirable to protect the moleculeagainst 5′→3′ exonuclease, the anti-microRNA molecule can have at leastone modified moiety and preferably at least two modified moieties at the5′ end of the molecule. The anti-microRNA molecule can also have atleast one and preferably at least two modified moieties between the 5′and 3′ end of the molecule to increase resistance of the molecule toendonucleases. In one embodiment, all of the moieties are nucleaseresistant.

In another embodiment, the anti-microRNA molecule comprises at least onemodified deoxyribonucleotide moiety. Suitable modifieddeoxyribonucleotide moieties are known in the art.

A suitable example of a modified deoxyribonucleotide moiety is aphosphorothioate deoxyribonucleotide moiety. See structure 1 in FIG. 1.An anti-microRNA molecule comprising more than one phosphorothioatedeoxyribonucleotide moiety is referred to as phosphorothioate (PS) DNA.See, for example, Eckstein, Antisense Nucleic Acids Drug Dev. 10,117-121 (2000).

Another suitable example of a modified deoxyribonucleotide moiety is anN′3-N′5 phosphoroamidate deoxyribonucleotide moiety. See structure 2 inFIG. 1. An oligonucleotide molecule comprising more than onephosphoroamidate deoxyribonucleotide moiety is referred to asphosphoroamidate (NP) DNA. See, for example, Gryaznov et al., J. Am.Chem. Soc. 116, 3143-3144 (1994).

In another embodiment, the molecule comprises at least one modifiedribonucleotide moiety. Suitable modified ribonucleotide moieties areknown in the art.

A suitable example of a modified ribonucleotide moiety is aribonucleotide moiety that is substituted at the 2′ position. Thesubstituents at the 2′ position may, for example, be a C₁ to C₄ alkylgroup. The C₁ to C₄ alkyl group may be saturated or unsaturated, andunbranched or branched. Some examples of C₁ to C₄ alkyl groups includeethyl, isopropyl, and allyl. The preferred C₁ to C₄ alkyl group ismethyl. See structure 3 in FIG. 1. An oligoribonucleotide moleculecomprising more than one ribonucleotide moeity that is substituted atthe 2′ position with a C₁ to C₄ alkyl group is referred to as a2′-O-(C₁-C₄ alkyl) RNA, e.g., 2′-O-methyl RNA (OMe RNA).

Another suitable example of a substituent at the 2′ position of amodified ribonucleotide moiety is a C₁ to C₄ alkoxy —C₁ to C₄ alkylgroup. The C₁ to C₄ alkoxy (alkyloxy) and C₁ to C₄ alkyl group maycomprise any of the alkyl groups described above. The preferred C₁ to C₄alkoxy —C₁ to C₄ alkyl group is methoxyethyl. See structure 4 in FIG. 1.An oligonucleotide molecule comprising more than one ribonucleotidemoiety that is substituted at the 2′ position with a C1 to C₄ alkoxy-C₁to C₄ alkyl group is referred to as a 2′-O—(C₁ to C₄ alkoxy-C₁ to C₄alkyl) RNA, e.g., 2′-O-methoxyethyl RNA (MOE RNA).

Another suitable example of a modified ribonucleotide moiety is aribonucleotide that has a methylene bridge between the 2′-oxygen atomand the 4′-carbon atom. See structure 5 in FIG. 1. Anoligoribonucleotide molecule comprising more than one ribonucleotidemoiety that has a methylene bridge between the 2′-oxygen atom and the4′-carbon atom is referred to as locked nucleic acid (LNA). See, forexample, Kurreck et al., Nucleic Acids Res. 30, 1911-1918 (2002);Elayadi et al., Curr. Opinion Invest. Drugs 2, 558-561 (2001); Ørum etal., Curr. Opinion Mol. Ther. 3, 239-243 (2001); Koshkin et al.,Tetrahedron 54, 3607-3630 (1998); Obika et al., Tetrahedron Lett. 39,5401-5404 (1998). Locked nucleic acids are commercially available fromProligo (Paris, France and Boulder, Colo., USA).

Another suitable example of a modified ribonucleotide moiety is aribonucleotide that is substituted at the 2′ position with fluoro group.A modified ribonucleotide moiety having a fluoro group at the 2′position is a 2′-fluororibonucleotide moiety. Such moieties are known inthe art. Molecules comprising more than one 2′-fluororibonucleotidemoiety are referred to herein as 2′-fluororibo nucleic acids (FANA). Seestructure 7 in FIG. 1. Damha et al., J. Am. Chem. Soc. 120, 12976-12977(1998).

In another embodiment, the anti-microRNA molecule comprises at least onebase bonded to an amino acid residue. Moieties that have at least onebase bonded to an amino acid residue will be referred to herein aspeptide nucleic acid (PNA) moieties. Such moieties are nucleaseresistance, and are known in the art. Molecules having more than one PNAmoiety are referred to as peptide nucleic acids. See structure 6 inFIG. 1. Nielson, Methods Enzymol. 313, 156-164 (1999); Elayadi, et al,id.; Braasch et al., Biochemistry 41, 4503-4509 (2002), Nielsen et al.,Science 254, 1497-1500 (1991).

The amino acids can be any amino acid, including natural or non-naturalamino acids. Naturally occurring amino acids include, for example, thetwenty most common amino acids normally found in proteins, i.e., alanine(Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine(Cys), glutamine (Glu), glutamic acid (Glu), glycine (Gly), histidine(His), isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan, (Trp), tyrosine (Tyr), and valine (Val).

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups. Some examples of alkyl amino acids includeα-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid,δ-aminovaleric acid, and ε-aminocaproic acid. Some examples of arylamino acids include ortho-, meta, and para-aminobenzoic acid. Someexamples of alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivative of a naturally occurringamino acid may, for example, include the addition or one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include hydroxyl, C₁-C₄ alkoxy, amino, methylamino,dimethylamino, nitro, halo (i.e., fluoro, chloro, bromo, or iodo), orbranched or unbranched C₁-C₄ alkyl, such as methyl, ethyl, n-propyl,isopropyl, butyl, isobutyl, or t-butyl.

Furthermore, other examples of non-naturally occurring amino acids whichare derivatives of naturally occurring amino acids include norvaline(Nva), norleucine (Nle), and hydroxyproline (Hyp).

The amino acids can be identical or different from one another. Basesare attached to the amino acid unit by molecular linkages. Examples oflinkages are methylene carbonyl, ethylene carbonyl and ethyl linkages.(Nielsen et al., Peptide Nucleic Acids-Protocols and Applications,Horizon Scientific Press, pages 1-19; Nielsen et al., Science 254:1497-1500.)

One example of a PNA moiety is N-(2-aminoethyl)-glycine. Furtherexamples of PNA moieties include cyclohexyl PNA, retro-inverso,phosphone, propionyl and aminoproline PNA.

PNA can be chemically synthesized by methods known in the art, e.g. bymodified Fmoc or tBoc peptide synthesis protocols. The PNA has manydesirable properties, including high melting temperatures (Tm), highbase-pairing specificity with nucleic acid and an uncharged molecularbackbone. Additionally, the PNA does not confer RNase H sensitivity onthe target RNA, and generally has good metabolic stability.

Peptide nucleic acids are also commercially available from AppliedBiosystems (Foster City, Calif., USA).

In another embodiment, the anti-microRNA molecule comprises at least onemorpholino phosphoroamidate nucleotide moiety. A morpholinophosphoroamidate nucleotide moiety is a modified moiety which isnuclease resistant. Such moieties are known in the art. Moleculescomprising more than one morpholino phosphoroamidate nucleotide moietyare referred to as morpholino (MF) nucleic acids. See structure 8 inFIG. 1. Heasman, Dev. Biol. 243, 209-214 (2002). Morpholonooligonucleotides are commercially available from Gene Tools LLC(Corvallis, Oreg., USA).

In another embodiment, the anti-microRNA molecule comprises at least onecyclohexene nucleotide moiety. A cyclohexene nucleotide moiety is amodified moiety which is nuclease resistant. Such moieties are known inthe art. Molecules comprising more than one cyclohexene nucleotidemoiety are referred to as cyclohexene nucleic acids (CeNA). Seestructure 10 in FIG. 1. Wang et al., J. Am. Chem. Soc. 122, 8595-8602(2000), Verbeure et al., Nucleic Acids Res. 29, 4941-4947 (2001).

In another embodiment, the anti-microRNA molecule comprises at least onetricyclo nucleotide moiety. A tricyclo nucleotide moiety is a modifiedmoiety which is nuclease resistant. Such moieties are known in the art.Steffens et al., J. Am. Chem. Soc. 119, 11548-11549 (1997), Renneberg etal., J. Am. Chem. Soc. 124, 5993-6002 (2002). Molecules comprising morethan one tricyclo nucleotide moiety are referred to as tricyclo nucleicacids (tcDNA). See structure 9 in FIG. 1.

In another embodiment, to increase nuclease resistance of theanti-microRNA molecules of the present invention to exonucleases,inverted nucleotide caps can be attached to the 5′ end, the 3′ end, orboth ends of the molecule. An inverted nucleotide cap refers to a 3′→5′sequence of nucleic acids attached to the anti-microRNA molecule at the5′ and/or the 3′ end. There is no limit to the maximum number ofnucleotides in the inverted cap just as long as it does not interferewith binding of the anti-microRNA molecule to its target microRNA. Anynucleotide can be used in the inverted nucleotide cap. Typically, theinverted nucleotide cap is one nucleotide in length. The nucleotide forthe inverted cap is generally thymine, but can be any nucleotide such asadenine, guanine, uracil, or cytosine.

Alternatively, an ethylene glycol compound and/or amino linkers can beattached to the either or both ends of the anti-microRNA molecule. Aminolinkers can also be used to increase nuclease resistance of theanti-microRNA molecules to endonucleases. The table below lists someexamples of amino linkers. The below listed amino linker arecommercially available from TriLink Biotechnologies, San Diego, Calif.

2′-Deoxycytidine-5-C6 Amino Linker (3′ Terminus) 2′-Deoxycytidine-5-C6Amino Linker (5′ or Internal) 3′ C3 Amino Linker 3′ C6 Amino Linker 3′C7 Amino Linker 5′ C12 Amino Linker 5′ C3 Amino Linker 5′ C6 AminoLinker C7 Internal Amino Linker Thymidine-5-C2 Amino Linker (5′ orInternal) Thymidine-5-C6 Amino Linker (3′ Terminus) Thymidine-5-C6 AminoLinker (Internal)

Chimeric anti-microRNA molecules containing a mixture of any of themoieties mentioned above are also known, and may be made by methodsknown, in the art. See, for example, references cited above, and Wang etal, Proc. Natl. Acad. Sci. USA 96, 13989-13994 (1999), Liang et al.,Eur. J. Biochem. 269, 5753-5758 (2002), Lok et al., Biochemistry 41,3457-3467 (2002), and Damha et al., J. Am. Chem. Soc. 120, 12976-12977(2002).

The molecules of the invention comprise at least ten contiguous,preferably at least thirteen contiguous, more preferably at leastfifteen contiguous, and even more preferably at least twenty contiguousbases that have the same sequence as a sequence of bases in any one ofthe anti-microRNA molecules shown in Tables 1-4. The anti-microRNAmolecules optimally comprise the entire sequence of any one of theanti-microRNA molecule sequences shown in Tables 1-4.

For the contiguous bases mentioned above, up to thirty percent of thebase pairs may be substituted by wobble base pairs. As used herein,wobble base pairs refers to either: i) substitution of a cytosine with auracil, or 2) the substitution of a adenine with a guanine, in thesequence of the anti-microRNA molecule. These wobble base pairs aregenerally referred to as UG or GU wobbles. Below is a table showing thenumber of contiguous bases and the maximum number of wobble base pairsin the anti-microRNA molecule:

Table for Number of Wobble Bases No. of Contiguous Bases 10 11 12 13 1415 16 17 18 Max. No. of 3 3 3 3 4 4 4 5 5 Wobble Base Pairs No. ofContiguous Bases 19 20 21 22 23 Max. No. of 5 6 6 6 6 Wobble Base Pairs

Further, up to ten percent, and preferably up to five percent of thecontiguous bases can be additions, deletions, mismatches or combinationsthereof. Additions refer to the insertion in the contiguous sequence ofany moiety described above comprising any one of the bases describedabove. Deletions refer to the removal of any moiety present in thecontiguous sequence. Mismatches refer to the substitution of one of themoieties comprising a base in the contiguous sequence with any of theabove described moieties comprising a different base.

The additions, deletions or mismatches can occur anywhere in thecontiguous sequence, for example, at either end of the contiguoussequence or within the contiguous sequence of the anti-microRNAmolecule. If the contiguous sequence is relatively short, such as fromabout ten to about 15 moieties in length, preferably the additions,deletions or mismatches occur at the end of the contiguous sequence. Ifthe contiguous sequence is relatively long, such as a minimum of sixteencontiguous sequences, then the additions, deletions, or mismatches canoccur anywhere in the contiguous sequence. Below is a table showing thenumber of contiguous bases and the maximum number of additions,deletions, mismatches or combinations thereof:

Table for Up to 10% No. of Contiguous Bases 10 11 12 13 14 15 16 17 18Max. No. of 1 1 1 1 1 1 1 1 1 Additions, Deletions and/or Mismatches No.of Contiguous Bases 19 20 21 22 23 Max. No. of 1 2 2 2 2 Additions,Deletions and/or Mismatches

Table for Up to 5% No. of Contiguous Bases 10 11 12 13 14 15 16 17 18Max. No. of 0 0 0 0 0 0 0 0 0 Additions, Deletions and/or Mismatches No.of Contiguous Bases 19 20 21 22 23 Max. No. of 0 1 1 1 1 Additions,Deletions and/or Mismatches

Furthermore, no more than fifty percent, and preferably no more thanthirty percent, of the contiguous moieties contain deoxyribonucleotidebackbone units. Below is a table showing the number of contiguous basesand the maximum number of deoxyribonucleotide backbone units:

Table for Fifty Percent Deoxyribonucleotide Backbone Units No. ofContiguous Bases 10 11 12 13 14 15 16 17 18 Max. No. of 5 5 6 6 7 7 8 89 Deoxyribonucleotide Backbone Units No. of Contiguous Bases 19 20 21 2223 Max. No. of Deoxyribonucleotide 9 10 10 11 11 Backbone Units

Table for Thirty Percent Deoxyribonucleotide Backbone Units No. ofContiguous Bases 10 11 12 13 14 15 16 17 18 Max. No. of 3 3 3 3 4 4 4 55 Deoxyribonucleotide Backbone Units No. of Contiguous Bases 19 20 21 2223 Max. No. of 5 6 6 6 6 Deoxyribonucleotide Backbone Units

The moiety in the anti-RNA molecule at the position corresponding toposition 11 of the microRNA is optionally non-complementary to amicroRNA. The moiety in the anti-microRNA molecule corresponding toposition 11 of the microRNA can be rendered non-complementary by anaddition, deletion or mismatch as described above.

In another embodiment, if the anti-microRNA molecule comprises onlyunmodified moieties, then the anti-microRNA molecules comprises at leastone base, in the at least ten contiguous bases, which isnon-complementary to the microRNA and/or comprises an invertednucleotide cap, ethylene glycol compound or an amino linker.

In yet another embodiment, if the at least ten contiguous bases in ananti-microRNA molecule is perfectly (i.e., 100%) complementary to tencontiguous bases in a microRNA, then the anti-microRNA molecule containsat least one modified moiety in the at least ten contiguous bases and/orcomprises an inverted nucleotide cap, ethylene glycol compound or anamino linker.

As stated above, the maximum length of the anti-microRNA molecule is 50moieties. Any number of moieties having any base sequence can be addedto the contiguous base sequence. The additional moieties can be added tothe 5′ end, the 3′ end, or to both ends of the contiguous sequence.

MicroRNA molecules are derived from genomic loci and are produced fromspecific microRNA genes. Mature microRNA molecules are processed fromprecursor transcripts that form local hairpin structures. The hairpinstructures are typically cleaved by an enzyme known as Dicer, whichgenerates one microRNA duplex. See Bartel, Cell 116, 281-297 (2004) fora review on microRNA molecules. The article by Bartel is herebyincorporated by reference.

Each strand of a microRNA is packaged in a microRNA ribonucleoproteincomplex (microRNP). A microRNP in, for example, humans, also includesthe proteins eIF2C2, the helicase Gemin3, and Gemin 4.

The sequence of bases in the anti-microRNA molecules of the presentinvention can be derived from a microRNA from any species e.g. such as afly (e.g., Drosophila melanogaster), a worm (e.g., C. elegans).Preferably the sequence of bases is found in mammals, especially humans(H. sapiens), mice (e.g., M. musculus), and rats (R. norvegicus).

The anti-microRNA molecule is preferably isolated, which means that itis essentially free of other nucleic acids. Essentially free from othernucleic acids means that it is at least 90%, preferably at least 95%and, more preferably, at least 98% free of other nucleic acids.

Preferably, the molecule is essentially pure, which means that themolecules is free not only of other nucleic acids, but also of othermaterials used in the synthesis of the molecule, such as, for example,enzymes used in the synthesis of the molecule. The molecule is at least90% free, preferably at least 95% free and, more preferably, at least98% free of such materials.

The anti-microRNA molecules of the present invention are capable ofinhibiting microRNP activity, preferable in a cell. Inhibiting microRNPactivity refers to the inhibition of cleavage of the microRNA's targetsequence or the repression of translation of the microRNA's targetsequence. The method comprises introducing into the cell asingle-stranded microRNA molecule.

Any anti-microRNA molecule can be used in the methods of the presentinvention, as long as the anti-microRNA is complementary, subject to therestrictions described above, to the microRNA present in the microRNP.Such anti-microRNAs include, for example, the anti-microRNA moleculesmentioned above (see Table 1-4), and the anti-microRNAs moleculesdescribed in international PCT application number WO 03/029459 A2, thesequences of which are incorporated herein by reference.

The invention also includes any one of the microRNA molecules having thesequences as shown in Table 2. The novel microRNA molecules in Table 2may optionally be modified as described above for anti-microRNAmolecules. The other microRNA molecules in Tables 1, 3 and 4 aremodified for increased nuclease resistance as described above foranti-microRNA molecules.

Utility

The anti-microRNA molecules and the microRNA molecules of the presentinvention have numerous in vivo, in vitro, and ex vivo applications.

For example, the anti-microRNA molecules and microRNA of the presentinvention may be used as a modulator of the expression of genes whichare at least partially complementary to the anti-microRNA molecules andmicroRNA. For example, if a particular microRNA is beneficial for thesurvival of a cell, an appropriate isolated microRNA of the presentinvention may be introduced into the cell to promote survival.Alternatively, if a particular microRNA is harmful (e.g., inducesapoptosis, induces cancer, etc.), an appropriate anti-microRNA moleculecan be introduced into the cell in order to inhibit the activity of themicroRNA and reduce the harm.

In addition, anti-microRNA molecules and/or microRNAs of the presentinvention can be introduced into a cell to study the function of themicroRNA. Any of the anti-microRNA molecules and/or microRNAs listedabove can be introduced into a cell for studying their function. Forexample, a microRNA in a cell can be inhibited with a suitableanti-microRNA molecule. The function of the microRNA can be inferred byobserving changes associated with inhibition of the microRNA in the cellin order to inhibit the activity of the microRNA and reduce the harm.

The cell can be any cell which expresses microRNA molecules, includingthe microRNA molecules listed herein. Alternatively, the cell can be anycell transfected with an expression vector containing the nucleotidesequence of a microRNA.

Examples of cells include, but are not limited to, endothelial cells,epithelial cells, leukocytes (e.g., T cells, B cells, neutrophils,macrophages, eosinophils, basophils, dendritic cells, natural killercells and monocytes), stem cells, hemopoietic cells, embryonic cells,cancer cells.

The anti-microRNA molecules or microRNAs can be introduced into a cellby any method known to those skilled in the art. Useful deliverysystems, include for example, liposomes and charged lipids. Liposomestypically encapsulate oligonucleotide molecules within their aqueouscenter. Charged lipids generally form lipid-oligonucleotide moleculecomplexes as a result of opposing charges.

These liposomes-oligonucleotide molecule complexes orlipid-oligonucleotide molecule complexes are usually internalized byendocytosis. The liposomes or charged lipids generally comprise helperlipids which disrupt the endosomal membrane and release theoligonucleotide molecules.

Other methods for introducing an anti-microRNA molecule or a microRNAinto a cell include use of delivery vehicles, such as dendrimers,biodegradable polymers, polymers of amino acids, polymers of sugars, andoligonucleotide-binding nanoparticles. In addition, pluoronic gel as adepot reservoir can be used to deliver the anti-microRNA oligonucleotidemolecules over a prolonged period. The above methods are described in,for example, Hughes et al., Drug Discovery Today 6, 303-315 (2001);Liang et al. Eur. J. Biochem. 269 5753-5758 (2002); and Becker et al.,In Antisense Technology in the Central Nervous System (Leslie, R. A.,Hunter, A. J. & Robertson, H. A., eds), pp. 147-157, Oxford UniversityPress.

Targeting of an anti-microRNA molecule or a microRNA to a particularcell can be performed by any method known to those skilled in the art.For example, the anti-microRNA molecule or microRNA can be conjugated toan antibody or ligand specifically recognized by receptors on the cell.

The sequences of microRNA and anti-microRNA molecules are shown inTables 1-4 below. Human sequences are indicated with the prefix “hsa.”Mouse sequences are indicated with the prefix “mmu.” Rat sequences areindicated with the prefix “rno.” C. elegan sequences are indicated withthe prefix “cel.” Drosophila sequences are indicated with the prefix“dme.”

TABLE 1 Human, Mouse and Rat microRNA and anti-microRNA sequences.microRNA name microRNA sequence (5′ to 3′)Anti-microRNA molecule sequence (5′ to 3′) hsa-miR-100SEQ ID NO. 1   AACCCGUAGAUCCGAACUUGUGSEQ ID NO. 307 CACAAGUUCGGAUCUACGGGUU hsa-miR-103SEQ ID NO. 2   AGCAGCAUUGUACAGGGCUAUGSEQ ID NO. 308 CAUAGCCCUGUACAAUGCUGCU hsa-miR-105-5pSEQ ID NO. 3   UCAAAUGCUCAGACUCCUGUGGSEQ ID NO. 309 CCACAGGAGUCUGAGCAUUUGA hsa-miR-106aSEQ ID NO. 4   AAAAGUGCUUACAGUGCAGGUASEQ ID NO. 310 UACCUGCACUGUAAGCACUUUU hsa-miR-106bSEQ ID NO. 5   UAAAGUGCUGACAGUGCAGAUASEQ ID NO. 311 UAUCUGCACUGUCAGCACUUUA hsa-miR-107SEQ ID NO. 6   AGCAGCAUUGUACAGGGCUAUCSEQ ID NO. 312 GAUAGCCCUGUACAAUGCUGCU hsa-miR-10bSEQ ID NO. 7   UACCCUGUAGAACCGAAUUUGUSEQ ID NO. 313 ACAAAUUCGGUUCUACAGGGUA hsa-miR-128bSEQ ID NO. 8   UCACAGUGAACCGGUCUCUUUCSEQ ID NO. 314 GAAAGAGACCGGUUCACUGUGA hsa-miR-130bSEQ ID NO. 9   CAGUGCAAUGAUGAAAGGGCAUSEQ ID NO. 315 AUGCCCUUUCAUCAUUGCACUG hsa-miR-140-3pSEQ ID NO. 10  UACCACAGGGUAGAACCACGGASEQ ID NO. 316 UCCGUGGUUCUACCCUGUGGUA hsa-miR-142-5pSEQ ID NO. 11  CCCAUAAAGUAGAAAGCACUACSEQ ID NO. 317 GUAGUGCUUUCUACUUUAUGGG hsa-miR-151-5pSEQ ID NO. 12  UCGAGGAGCUCACAGUCUAGUASEQ ID NO. 318 UACUAGACUGUGAGCUCCUCGA hsa-miR-155SEQ ID NO. 13  UUAAUGCUAAUCGUGAUAGGGGSEQ ID NO. 319 CCCCUAUCACGAUUAGCAUUAA hsa-miR-181aSEQ ID NO. 14  AACAUUCAACGCUGUCGGUGAGSEQ ID NO. 320 CUCACCGACAGCGUUGAAUGUU hsa-miR-181bSEQ ID NO. 15  AACAUUCAUUGCUGUCGGUGGGSEQ ID NO. 321 CCCACCGACAGCAAUGAAUGUU hsa-miR-181cSEQ ID NO. 16  AACAUUCAACCUGUCGGUGAGUSEQ ID NO. 322 ACUCACCGACAGGUUGAAUGUU hsa-miR-182SEQ ID NO. 17  UUUGGCAAUGGUAGAACUCACASEQ ID NO. 323 UGUGAGUUCUACCAUUGCCAAA hsa-miR-183SEQ ID NO. 18  UAUGGCACUGGUAGAAUUCACUSEQ ID NO. 324 AGUGAAUUCUACCAGUGCCAUA hsa-miR-184SEQ ID NO. 19  UGGACGGAGAACUGAUAAGGGUSEQ ID NO. 325 ACCCUUAUCAGUUCUCCGUCCA hsa-miR-185SEQ ID NO. 20  UGGAGAGAAAGGCAGUUCCUGASEQ ID NO. 326 UCAGGAACUGCCUUUCUCUCCA hsa-miR-186SEQ ID NO. 21  CAAAGAAUUCUCCUUUUGGGCUSEQ ID NO. 327 AGCCCAAAAGGAGAAUUCUUUG hsa-miR-187SEQ ID NO. 22  UCGUGUCUUGUGUUGCAGCCGGSEQ ID NO. 328 CCGGCUGCAACACAAGACACGA hsa-miR-188-3pSEQ ID NO. 23  CUCCCACAUGCAGGGUUUGCAGSEQ ID NO. 329 CUGCAAACCCUGCAUGUGGGAG hsa-miR-188-5pSEQ ID NO. 24  CAUCCCUUGCAUGGUGGAGGGUSEQ ID NO. 330 ACCCUCCACCAUGCAAGGGAUG hsa-miR-189SEQ ID NO. 25  GUGCCUACUGAGCUGAUAUCAGSEQ ID NO. 331 CUGAUAUCAGCUCAGUAGGCAC hsa-miR-190SEQ ID NO. 26  UGAUAUGUUUGAUAUAUUAGGUSEQ ID NO. 332 ACCUAAUAUAUCAAACAUAUCA hsa-miR-191SEQ ID NO. 27  CAACGGAAUCCCAAAAGCAGCUSEQ ID NO. 333 AGCUGCUUUUGGGAUUCCGUUG hsa-miR-192SEQ ID NO. 28  CUGACCUAUGAAUUGACAGCCASEQ ID NO. 334 UGGCUGUCAAUUCAUAGGUCAG hsa-miR-193-3pSEQ ID NO. 29  AACUGGCCUACAAAGUCCCAGUSEQ ID NO. 335 ACUGGGACUUUGUAGGCCAGUU hsa-miR-193-5pSEQ ID NO. 30  UGGGUCUUUGCGGGCAAGAUGASEQ ID NO. 336 UCAUCUUGCCCGCAAAGACCCA hsa-miR-194SEQ ID NO. 31  UGUAACAGCAACUCCAUGUGGASEQ ID NO. 337 UCCACAUGGAGUUGCUGUUACA hsa-miR-195SEQ ID NO. 32  UAGCAGCACAGAAAUAUUGGCASEQ ID NO. 338 UGCCAAUAUUUCUGUGCUGCUA hsa-miR-196SEQ ID NO. 33  UAGGUAGUUUCAUGUUGUUGGGSEQ ID NO. 339 CCCAACAACAUGAAACUACCUA hsa-miR-197SEQ ID NO. 34  UUCACCACCUUCUCCACCCAGCSEQ ID NO. 340 GCUGGGUGGAGAAGGUGGUGAA hsa-miR-198SEQ ID NO. 35  GGUCCAGAGGGGAGAUAGGUUCSEQ ID NO. 341 GAACCUAUCUCCCCUCUGGACC hsa-miR-199a-SEQ ID NO. 36  ACAGUAGUCUGCACAUUGGUUASEQ ID NO. 342 UAACCAAUGUGCAGACUACUGU 3p hsa-miR-199a-SEQ ID NO. 37  CCCAGUGUUCAGACUACCUGUUSEQ ID NO. 343 AACAGGUAGUCUGAACACUGGG 5p hsa-miR-199bSEQ ID NO. 38  CCCAGUGUUUAGACUAUCUGUUSEQ ID NO. 344 AACAGAUAGUCUAAACACUGGG hsa-miR-200aSEQ ID NO. 39  UAACACUGUCUGGUAACGAUGUSEQ ID NO. 345 ACAUCGUUACCAGACAGUGUUA hsa-miR-200bSEQ ID NO. 40  CUCUAAUACUGCCUGGUAAUGASEQ ID NO. 346 UCAUUACCAGGCAGUAUUAGAG hsa-miR-200cSEQ ID NO. 41  AAUACUGCCGGGUAAUGAUGGASEQ ID NO. 347 UCCAUCAUUACCCGGCAGUAUU hsa-miR-203SEQ ID NO. 42  GUGAAAUGUUUAGGACCACUAGSEQ ID NO. 348 CUAGUGGUCCUAAACAUUUCAC hsa-miR-204SEQ ID NO. 43  UUCCCUUUGUCAUCCUAUGCCUSEQ ID NO. 349 AGGCAUAGGAUGACAAAGGGAA hsa-miR-205SEQ ID NO. 44  UCCUUCAUUCCACCGGAGUCUGSEQ ID NO. 350 CAGACUCCGGUGGAAUGAAGGA hsa-miR-206SEQ ID NO. 45  UGGAAUGUAAGGAAGUGUGUGGSEQ ID NO. 351 CCACACACUUCCUUACAUUCCA hsa-miR-208SEQ ID NO. 46  AUAAGACGAGCAAAAAGCUUGUSEQ ID NO. 352 ACAAGCUUUUUGCUCGUCUUAU hsa-miR-210SEQ ID NO. 47  CUGUGCGUGUGACAGCGGCUGASEQ ID NO. 353 UCAGCCGCUGUCACACGCACAG hsa-miR-211SEQ ID NO. 48  UUCCCUUUGUCAUCCUUCGCCUSEQ ID NO. 354 AGGCGAAGGAUGACAAAGGGAA hsa-miR-212SEQ ID NO. 49  UAACAGUCUCCAGUCACGGCCASEQ ID NO. 355 UGGCCGUGACUGGAGACUGUUA hsa-miR-213SEQ ID NO. 50  ACCAUCGACCGUUGAUUGUACCSEQ ID NO. 356 GGUACAAUCAACGGUCGAUGGU hsa-miR-214SEQ ID NO. 51  ACAGCAGGCACAGACAGGCAGUSEQ ID NO. 357 ACUGCCUGUCUGUGCCUGCUGU hsa-miR-215SEQ ID NO. 52  AUGACCUAUGAAUUGACAGACASEQ ID NO. 358 UGUCUGUCAAUUCAUAGGUCAU hsa-miR-216SEQ ID NO. 53  UAAUCUCAGCUGGCAACUGUGASEQ ID NO. 359 UCACAGUUGCCAGCUGAGAUUA hsa-miR-217SEQ ID NO. 54  UACUGCAUCAGGAACUGAUUGGSEQ ID NO. 360 CCAAUCAGUUCCUGAUGCAGUA hsa-miR-218SEQ ID NO. 55  UUGUGCUUGAUCUAACCAUGUGSEQ ID NO. 361 CACAUGGUUAGAUCAAGCACAA hsa-miR-219SEQ ID NO. 56  UGAUUGUCCAAACGCAAUUCUUSEQ ID NO. 362 AAGAAUUGCGUUUGGACAAUCA hsa-miR-220SEQ ID NO. 57  CCACACCGUAUCUGACACUUUGSEQ ID NO. 363 CAAAGUGUCAGAUACGGUGUGG hsa-miR-221SEQ ID NO. 58  AGCUACAUUGUCUGCUGGGUUUSEQ ID NO. 364 AAACCCAGCAGACAAUGUAGCU hsa-miR-222SEQ ID NO. 59  AGCUACAUCUGGCUACUGGGUCSEQ ID NO. 365 GACCCAGUAGCCAGAUGUAGCU hsa-miR-223SEQ ID NO. 60  UGUCAGUUUGUCAAAUACCCCASEQ ID NO. 366 UGGGGUAUUUGACAAACUGACA hsa-miR-224SEQ ID NO. 61  CAAGUCACUAGUGGUUCCGUUUSEQ ID NO. 367 AAACGGAACCACUAGUGACUUG hsa-miR-28-5pSEQ ID NO. 62  AAGGAGCUCACAGUCUAUUGAGSEQ ID NO. 368 CUCAAUAGACUGUGAGCUCCUU hsa-miR-290SEQ ID NO. 63  CUCAAACUGUGGGGGCACUUUCSEQ ID NO. 369 GAAAGUGCCCCCACAGUUUGAG hsa-miR-296SEQ ID NO. 64  AGGGCCCCCCCUCAAUCCUGUUSEQ ID NO. 370 AACAGGAUUGAGGGGGGGCCCU hsa-miR-299SEQ ID NO. 65  UGGUUUACCGUCCCACAUACAUSEQ ID NO. 371 AUGUAUGUGGGACGGUAAACCA hsa-miR-301SEQ ID NO. 66  CAGUGCAAUAGUAUUGUCAAAGSEQ ID NO. 372 CUUUGACAAUACUAUUGCACUG hsa-miR-302SEQ ID NO. 67  UAAGUGCUUCCAUGUUUUGGUGSEQ ID NO. 373 CACCAAAACAUGGAAGCACUUA hsa-miR-30eSEQ ID NO. 68  UGUAAACAUCCUUGACUGGAAGSEQ ID NO. 374 CUUCCAGUCAAGGAUGUUUACA hsa-miR-320SEQ ID NO. 69  AAAAGCUGGGUUGAGAGGGCGASEQ ID NO. 375 UCGCCCUCUCAACCCAGCUUUU hsa-miR-321SEQ ID NO. 70  UAAGCCAGGGAUUGUGGGUUCGSEQ ID NO. 376 CGAACCCACAAUCCCUGGCUUA hsa-miR-322SEQ ID NO. 71  AAACAUGAAUUGCUGCUGUAUCSEQ ID NO. 377 GAUACAGCAGCAAUUCAUGUUU hsa-miR-323SEQ ID NO. 72  GCACAUUACACGGUCGACCUCUSEQ ID NO. 378 AGAGGUCGACCGUGUAAUGUGC hsa-miR-324-3pSEQ ID NO. 73  CCACUGCCCCAGGUGCUGCUGGSEQ ID NO. 379 CCAGCAGCACCUGGGGCAGUGG hsa-miR-324-5pSEQ ID NO. 74  CGCAUCCCCUAGGGCAUUGGUGSEQ ID NO. 380 CACCAAUGCCCUAGGGGAUGCG hsa-miR-326SEQ ID NO. 75  CCUCUGGGCCCUUCCUCCAGCCSEQ ID NO. 381 GGCUGGAGGAAGGGCCCAGAGG hsa-miR-328SEQ ID NO. 76  CUGGCCCUCUCUGCCCUUCCGUSEQ ID NO. 382 ACGGAAGGGCAGAGAGGGCCAG hsa-miR-329SEQ ID NO. 77  AACACACCCAGCUAACCUUUUUSEQ ID NO. 383 AAAAAGGUUAGCUGGGUGUGUU hsa-miR-34aSEQ ID NO. 78  UGGCAGUGUCUUAGCUGGUUGUSEQ ID NO. 384 ACAACCAGCUAAGACACUGCCA hsa-miR-34bSEQ ID NO. 79  AGGCAGUGUCAUUAGCUGAUUGSEQ ID NO. 385 CAAUCAGCUAAUGACACUGCCU hsa-miR-34cSEQ ID NO. 80  AGGCAGUGUAGUUAGCUGAUUGSEQ ID NO. 386 CAAUCAGCUAACUACACUGCCU hsa-miR-92SEQ ID NO. 81  UAUUGCACUUGUCCCGGCCUGUSEQ ID NO. 387 ACAGGCCGGGACAAGUGCAAUA hsa-miR-93SEQ ID NO. 82  AAAGUGCUGUUCGUGCAGGUAGSEQ ID NO. 388 CUACCUGCACGAACAGCACUUU hsa-miR-95SEQ ID NO. 83  UUCAACGGGUAUUUAUUGAGCASEQ ID NO. 389 UGCUCAAUAAAUACCCGUUGAA hsa-miR-96SEQ ID NO. 84  UUUGGCACUAGCACAUUUUUGCSEQ ID NO. 390 GCAAAAAUGUGCUAGUGCCAAA hsa-miR-98SEQ ID NO. 85  UGAGGUAGUAAGUUGUAUUGUUSEQ ID NO. 391 AACAAUACAACUUACUACCUCA mmu-miR-106aSEQ ID NO. 86  CAAAGUGCUAACAGUGCAGGUASEQ ID NO. 392 UACCUGCACUGUUAGCACUUUG mmu-miR-10bSEQ ID NO. 87  CCCUGUAGAACCGAAUUUGUGUSEQ ID NO. 393 ACACAAAUUCGGUUCUACAGGG mmu-miR-135bSEQ ID NO. 88  UAUGGCUUUUCAUUCCUAUGUGSEQ ID NO. 394 CACAUAGGAAUGAAAAGCCAUA mmu-miR-148bSEQ ID NO. 89  UCAGUGCAUCACAGAACUUUGUSEQ ID NO. 395 ACAAAGUUCUGUGAUGCACUGA mmu-miR-151-3pSEQ ID NO. 90  CUAGACUGAGGCUCCUUGAGGASEQ ID NO. 396 UCCUCAAGGAGCCUCAGUCUAG mmu-miR-155SEQ ID NO. 91  UUAAUGCUAAUUGUGAUAGGGGSEQ ID NO. 397 CCCCUAUCACAAUUAGCAUUAA mmu-miR-199bSEQ ID NO. 92  CCCAGUGUUUAGACUACCUGUUSEQ ID NO. 398 AACAGGUAGUCUAAACACUGGG mmu-miR-200bSEQ ID NO. 93  UAAUACUGCCUGGUAAUGAUGASEQ ID NO. 399 UCAUCAUUACCAGGCAGUAUUA mmu-miR-203SEQ ID NO. 94  UGAAAUGUUUAGGACCACUAGASEQ ID NO. 400 UCUAGUGGUCCUAAACAUUUCA mmu-miR-211SEQ ID NO. 95  UUCCCUUUGUCAUCCUUUGCCUSEQ ID NO. 401 AGGCAAAGGAUGACAAAGGGAA mmu-miR-217SEQ ID NO. 96  UACUGCAUCAGGAACUGACUGGSEQ ID NO. 402 CCAGUCAGUUCCUGAUGCAGUA mmu-miR-224SEQ ID NO. 97  UAAGUCACUAGUGGUUCCGUUUSEQ ID NO. 403 AAACGGAACCACUAGUGACUUA mmu-miR-28-3pSEQ ID NO. 98  CACUAGAUUGUGAGCUGCUGGASEQ ID NO. 404 UCCAGCAGCUCACAAUCUAGUG mmu-miR-290SEQ ID NO. 99  CUCAAACUAUGGGGGCACUUUUSEQ ID NO. 405 AAAAGUGCCCCCAUAGUUUGAG mmu-miR-291-3pSEQ ID NO. 100 AAAGUGCUUCCACUUUGUGUGCSEQ ID NO. 406 GCACACAAAGUGGAAGCACUUU mmu-miR-291-5pSEQ ID NO. 101 CAUCAAAGUGGAGGCCCUCUCUSEQ ID NO. 407 AGAGAGGGCCUCCACUUUGAUG mmu-miR-292-3pSEQ ID NO. 102 AAGUGCCGCCAGGUUUUGAGUGSEQ ID NO. 408 CACUCAAAACCUGGCGGCACUU mmu-miR-292-5pSEQ ID NO. 103 ACUCAAACUGGGGGCUCUUUUGSEQ ID NO. 409 CAAAAGAGCCCCCAGUUUGAGU mmu-miR-293SEQ ID NO. 104 AGUGCCGCAGAGUUUGUAGUGUSEQ ID NO. 410 ACACUACAAACUCUGCGGCACU mmu-miR-294SEQ ID NO. 105 AAAGUGCUUCCCUUUUGUGUGUSEQ ID NO. 411 ACACACAAAAGGGAAGCACUUU mmu-miR-295SEQ ID NO. 106 AAAGUGCUACUACUUUUGAGUCSEQ ID NO. 412 GACUCAAAAGUAGUAGCACUUU mmu-miR-297SEQ ID NO. 107 AUGUAUGUGUGCAUGUGCAUGUSEQ ID NO. 413 ACAUGCACAUGCACACAUACAU mmu-miR-298SEQ ID NO. 108 GGCAGAGGAGGGCUGUUCUUCCSEQ ID NO. 414 GGAAGAACAGCCCUCCUCUGCC mmu-miR-300SEQ ID NO. 109 UAUGCAAGGGCAAGCUCUCUUCSEQ ID NO. 415 GAAGAGAGCUUGCCCUUGCAUA mmu-miR-31SEQ ID NO. 110 AGGCAAGAUGCUGGCAUAGCUGSEQ ID NO. 416 CAGCUAUGCCAGCAUCUUGCCU mmu-miR-322SEQ ID NO. 111 AAACAUGAAGCGCUGCAACACCSEQ ID NO. 417 GGUGUUGCAGCGCUUCAUGUUU mmu-miR-325SEQ ID NO. 112 CCUAGUAGGUGCUCAGUAAGUGSEQ ID NO. 418 CACUUACUGAGCACCUACUAGG mmu-miR-326SEQ ID NO. 113 CCUCUGGGCCCUUCCUCCAGUCSEQ ID NO. 419 GACUGGAGGAAGGGCCCAGAGG mmu-miR-330SEQ ID NO. 114 GCAAAGCACAGGGCCUGCAGAGSEQ ID NO. 420 CUCUGCAGGCCCUGUGCUUUGC mmu-miR-331SEQ ID NO. 115 GCCCCUGGGCCUAUCCUAGAACSEQ ID NO. 421 GUUCUAGGAUAGGCCCAGGGGC mmu-miR-337SEQ ID NO. 116 UUCAGCUCCUAUAUGAUGCCUUSEQ ID NO. 422 AAGGCAUCAUAUAGGAGCUGAA mmu-miR-338SEQ ID NO. 117 UCCAGCAUCAGUGAUUUUGUUGSEQ ID NO. 423 CAACAAAAUCACUGAUGCUGGA mmu-miR-339SEQ ID NO. 118 UCCCUGUCCUCCAGGAGCUCACSEQ ID NO. 424 GUGAGCUCCUGGAGGACAGGGA mmu-miR-340SEQ ID NO. 119 UCCGUCUCAGUUACUUUAUAGCSEQ ID NO. 425 GCUAUAAAGUAACUGAGACGGA mmu-miR-341SEQ ID NO. 120 UCGAUCGGUCGGUCGGUCAGUCSEQ ID NO. 426 GACUGACCGACCGACCGAUCGA mmu-miR-342SEQ ID NO. 121 UCUCACACAGAAAUCGCACCCGSEQ ID NO. 427 CGGGUGCGAUUUCUGUGUGAGA mmu-miR-344SEQ ID NO. 122 UGAUCUAGCCAAAGCCUGACUGSEQ ID NO. 428 CAGUCAGGCUUUGGCUAGAUCA mmu-miR-345SEQ ID NO. 123 UGCUGACCCCUAGUCCAGUGCUSEQ ID NO. 429 AGCACUGGACUAGGGGUCAGCA mmu-miR-346SEQ ID NO. 124 UGUCUGCCCGAGUGCCUGCCUCSEQ ID NO. 430 GAGGCAGGCACUCGGGCAGACA mmu-miR-34bSEQ ID NO. 125 UAGGCAGUGUAAUUAGCUGAUUSEQ ID NO. 431 AAUCAGCUAAUUACACUGCCUA mmu-miR-350SEQ ID NO. 126 UUCACAAAGCCCAUACACUUUCSEQ ID NO. 432 GAAAGUGUAUGGGCUUUGUGAA mmu-miR-351SEQ ID NO. 127 UCCCUGAGGAGCCCUUUGAGCCSEQ ID NO. 433 GGCUCAAAGGGCUCCUCAGGGA mmu-miR-7bSEQ ID NO. 128 UGGAAGACUUGUGAUUUUGUUGSEQ ID NO. 434 CAACAAAAUCACAAGUCUUCCA mmu-miR-92SEQ ID NO. 129 UAUUGCACUUGUCCCGGCCUGASEQ ID NO. 435 UCAGGCCGGGACAAGUGCAAUA mmu-miR-93SEQ ID NO. 130 CAAAGUGCUGUUCGUGCAGGUASEQ ID NO. 436 UACCUGCACGAACAGCACUUUG rno-miR-327SEQ ID NO. 131 CCUUGAGGGGCAUGAGGGUAGUSEQ ID NO. 437 ACUACCCUCAUGCCCCUCAAGG rno-miR-333SEQ ID NO. 132 GUGGUGUGCUAGUUACUUUUGGSEQ ID NO. 438 CCAAAAGUAACUAGCACACCAC rno-miR-335SEQ ID NO. 133 UCAAGAGCAAUAACGAAAAAUGSEQ ID NO. 439 CAUUUUUCGUUAUUGCUCUUGA rno-miR-336SEQ ID NO. 134 UCACCCUUCCAUAUCUAGUCUCSEQ ID NO. 440 GAGACUAGAUAUGGAAGGGUGA rno-miR-343SEQ ID NO. 135 UCUCCCUCCGUGUGCCCAGUAUSEQ ID NO. 441 AUACUGGGCACACGGAGGGAGA rno-miR-347SEQ ID NO. 136 UGUCCCUCUGGGUCGCCCAGCUSEQ ID NO. 442 AGCUGGGCGACCCAGAGGGACA rno-miR-349SEQ ID NO. 137 CAGCCCUGCUGUCUUAACCUCUSEQ ID NO. 443 AGAGGUUAAGACAGCAGGGCUG rno-miR-352SEQ ID NO. 138 AGAGUAGUAGGUUGCAUAGUACSEQ ID NO. 444 GUACUAUGCAACCUACUACUCU

TABLE 2 Novel Human microRNA and anti-microRNA sequences. microRNA namemicroRNA sequence (5′ to 3′) Anti-microRNA molecule sequence (5′ to 3′)hsa-miR-361 SEQ ID NO. 139 UUAUCAGAAUCUCCAGGGGUACSEQ ID NO. 445 GUACCCCUGGAGAUUCUGAUAA hsa-miR-362SEQ ID NO. 140 AAUCCUUGGAACCUAGGUGUGASEQ ID NO. 446 UCACACCUAGGUUCCAAGGAUU hsa-miR-363SEQ ID NO. 141 AUUGCACGGUAUCCAUCUGUAASEQ ID NO. 447 UUACAGAUGGAUACCGUGCAAU hsa-miR-364SEQ ID NO. 142 CGGCGGGGACGGCGAUUGGUCCSEQ ID NO. 448 GGACCAAUCGCCGUCCCCGCCG hsa-miR-365SEQ ID NO. 143 UAAUGCCCCUAAAAAUCCUUAUSEQ ID NO. 449 AUAAGGAUUUUUAGGGGCAUUA hsa-miR-366SEQ ID NO. 144 UAACUGGUUGAACAACUGAACCSEQ ID NO. 450 GGUUCAGUUGUUCAACCAGUUA

TABLE 3 C. elegans microRNA and anti-microRNA sequences. microRNA namemicroRNA sequence (5′ to 3′) Anti-microRNA molecule sequence (5′ to 3′)Cel-let-7 SEQ ID NO. 145 UGAGGUAGUAGGUUGUAUAGUUSEQ ID NO. 451 AACUAUACAACCUACUACCUCA Cel-lin-4SEQ ID NO. 146 UCCCUGAGACCUCAAGUGUGAGSEQ ID NO. 452 CUCACACUUGAGGUCUCAGGGA Cel-miR-1SEQ ID NO. 147 UGGAAUGUAAAGAAGUAUGUAGSEQ ID NO. 453 CUACAUACUUCUUUACAUUCCA Cel-miR-2SEQ ID NO. 148 UAUCACAGCCAGCUUUGAUGUGSEQ ID NO. 454 CACAUCAAAGCUGGCUGUGAUA Cel-miR-34SEQ ID NO. 149 AGGCAGUGUGGUUAGCUGGUUGSEQ ID NO. 455 CAACCAGCUAACCACACUGCCU Cel-miR-35SEQ ID NO. 150 UCACCGGGUGGAAACUAGCAGUSEQ ID NO. 456 ACUGCUAGUUUCCACCCGGUGA Cel-miR-36SEQ ID NO. 151 UCACCGGGUGAAAAUUCGCAUGSEQ ID NO. 457 CAUGCGAAUUUUCACCCGGUGA Cel-miR-37SEQ ID NO. 152 UCACCGGGUGAACACUUGCAGUSEQ ID NO. 458 ACUGCAAGUGUUCACCCGGUGA Cel-miR-38SEQ ID NO. 153 UCACCGGGAGAAAAACUGGAGUSEQ ID NO. 459 ACUCCAGUUUUUCUCCCGGUGA Cel-miR-39SEQ ID NO. 154 UCACCGGGUGUAAAUCAGCUUGSEQ ID NO. 460 CAAGCUGAUUUACACCCGGUGA Cel-miR-40SEQ ID NO. 155 UCACCGGGUGUACAUCAGCUAASEQ ID NO. 461 UUAGCUGAUGUACACCCGGUGA Cel-miR-41SEQ ID NO. 156 UCACCGGGUGAAAAAUCACCUASEQ ID NO. 462 UAGGUGAUUUUUCACCCGGUGA Cel-miR-42SEQ ID NO. 157 CACCGGGUUAACAUCUACAGAGSEQ ID NO. 463 CUCUGUAGAUGUUAACCCGGUG Cel-miR-43SEQ ID NO. 158 UAUCACAGUUUACUUGCUGUCGSEQ ID NO. 464 CGACAGCAAGUAAACUGUGAUA Cel-miR-44SEQ ID NO. 159 UGACUAGAGACACAUUCAGCUUSEQ ID NO. 465 AAGCUGAAUGUGUCUCUAGUCA Cel-miR-45SEQ ID NO. 160 UGACUAGAGACACAUUCAGCUUSEQ ID NO. 466 AAGCUGAAUGUGUCUCUAGUCA Cel-miR-46SEQ ID NO. 161 UGUCAUGGAGUCGCUCUCUUCASEQ ID NO. 467 UGAAGAGAGCGACUCCAUGACA Cel-miR-47SEQ ID NO. 162 UGUCAUGGAGGCGCUCUCUUCASEQ ID NO. 468 UGAAGAGAGCGCCUCCAUGACA Cel-miR-48SEQ ID NO. 163 UGAGGUAGGCUCAGUAGAUGCGSEQ ID NO. 469 CGCAUCUACUGAGCCUACCUCA Cel-miR-49SEQ ID NO. 164 AAGCACCACGAGAAGCUGCAGASEQ ID NO. 470 UCUGCAGCUUCUCGUGGUGCUU Cel-miR-50SEQ ID NO. 165 UGAUAUGUCUGGUAUUCUUGGGSEQ ID NO. 471 CCCAAGAAUACCAGACAUAUCA Cel-miR-51SEQ ID NO. 166 UACCCGUAGCUCCUAUCCAUGUSEQ ID NO. 472 ACAUGGAUAGGAGCUACGGGUA Cel-miR-52SEQ ID NO. 167 CACCCGUACAUAUGUUUCCGUGSEQ ID NO. 473 CACGGAAACAUAUGUACGGGUG Cel-miR-53SEQ ID NO. 168 CACCCGUACAUUUGUUUCCGUGSEQ ID NO. 474 CACGGAAACAAAUGUACGGGUG Cel-miR-54SEQ ID NO. 169 UACCCGUAAUCUUCAUAAUCCGSEQ ID NO. 475 CGGAUUAUGAAGAUUACGGGUA Cel-miR-55SEQ ID NO. 170 UACCCGUAUAAGUUUCUGCUGASEQ ID NO. 476 UCAGCAGAAACUUAUACGGGUA Cel-miR-56SEQ ID NO. 171 UACCCGUAAUGUUUCCGCUGAGSEQ ID NO. 477 CUCAGCGGAAACAUUACGGGUA Cel-miR-57SEQ ID NO. 172 UACCCUGUAGAUCGAGCUGUGUSEQ ID NO. 478 ACACAGCUCGAUCUACAGGGUA Cel-miR-58SEQ ID NO. 173 UGAGAUCGUUCAGUACGGCAAUSEQ ID NO. 479 AUUGCCGUACUGAACGAUCUCA Cel-miR-59SEQ ID NO. 174 UCGAAUCGUUUAUCAGGAUGAUSEQ ID NO. 480 AUCAUCCUGAUAAACGAUUCGA Cel-miR-60SEQ ID NO. 175 UAUUAUGCACAUUUUCUAGUUCSEQ ID NO. 481 GAACUAGAAAAUGUGCAUAAUA Cel-miR-61SEQ ID NO. 176 UGACUAGAACCGUUACUCAUCUSEQ ID NO. 482 AGAUGAGUAACGGUUCUAGUCA Cel-miR-62SEQ ID NO. 177 UGAUAUGUAAUCUAGCUUACAGSEQ ID NO. 483 CUGUAAGCUAGAUUACAUAUCA Cel-miR-63SEQ ID NO. 178 AUGACACUGAAGCGAGUUGGAASEQ ID NO. 484 UUCCAACUCGCUUCAGUGUCAU Cel-miR-64SEQ ID NO. 179 UAUGACACUGAAGCGUUACCGASEQ ID NO. 485 UCGGUAACGCUUCAGUGUCAUA Cel-miR-65SEQ ID NO. 180 UAUGACACUGAAGCGUAACCGASEQ ID NO. 486 UCGGUUACGCUUCAGUGUCAUA Cel-miR-66SEQ ID NO. 181 CAUGACACUGAUUAGGGAUGUGSEQ ID NO. 487 CACAUCCCUAAUCAGUGUCAUG Cel-miR-67SEQ ID NO. 182 UCACAACCUCCUAGAAAGAGUASEQ ID NO. 488 UACUCUUUCUAGGAGGUUGUGA Cel-miR-68SEQ ID NO. 183 UCGAAGACUCAAAAGUGUAGACSEQ ID NO. 489 GUCUACACUUUUGAGUCUUCGA Cel-miR-69SEQ ID NO. 184 UCGAAAAUUAAAAAGUGUAGAASEQ ID NO. 490 UUCUACACUUUUUAAUUUUCGA Cel-miR-70SEQ ID NO. 185 UAAUACGUCGUUGGUGUUUCCASEQ ID NO. 491 UGGAAACACCAACGACGUAUUA Cel-miR-71SEQ ID NO. 186 UGAAAGACAUGGGUAGUGAACGSEQ ID NO. 492 CGUUCACUACCCAUGUCUUUCA Cel-miR-72SEQ ID NO. 187 AGGCAAGAUGUUGGCAUAGCUGSEQ ID NO. 493 CAGCUAUGCCAACAUCUUGCCU Cel-miR-73SEQ ID NO. 188 UGGCAAGAUGUAGGCAGUUCAGSEQ ID NO. 494 CUGAACUGCCUACAUCUUGCCA Cel-miR-74SEQ ID NO. 189 UGGCAAGAAAUGGCAGUCUACASEQ ID NO. 495 UGUAGACUGCCAUUUCUUGCCA Cel-miR-75SEQ ID NO. 190 UUAAAGCUACCAACCGGCUUCASEQ ID NO. 496 UGAAGCCGGUUGGUAGCUUUAA Cel-miR-76SEQ ID NO. 191 UUCGUUGUUGAUGAAGCCUUGASEQ ID NO. 497 UCAAGGCUUCAUCAACAACGAA Cel-miR-77SEQ ID NO. 192 UUCAUCAGGCCAUAGCUGUCCASEQ ID NO. 498 UGGACAGCUAUGGCCUGAUGAA Cel-miR-78SEQ ID NO. 193 UGGAGGCCUGGUUGUUUGUGCUSEQ ID NO. 499 AGCACAAACAACCAGGCCUCCA Cel-miR-79SEQ ID NO. 194 AUAAAGCUAGGUUACCAAAGCUSEQ ID NO. 500 AGCUUUGGUAACCUAGCUUUAU Cel-miR-227SEQ ID NO. 195 AGCUUUCGACAUGAUUCUGAACSEQ ID NO. 501 GUUCAGAAUCAUGUCGAAAGCU Cel-miR-80SEQ ID NO. 196 UGAGAUCAUUAGUUGAAAGCCGSEQ ID NO. 502 CGGCUUUCAACUAAUGAUCUCA Cel-miR-81SEQ ID NO. 197 UGAGAUCAUCGUGAAAGCUAGUSEQ ID NO. 503 ACUAGCUUUCACGAUGAUCUCA Cel-miR-82SEQ ID NO. 198 UGAGAUCAUCGUGAAAGCCAGUSEQ ID NO. 504 ACUGGCUUUCACGAUGAUCUCA Cel-miR-83SEQ ID NO. 199 UAGCACCAUAUAAAUUCAGUAASEQ ID NO. 505 UUACUGAAUUUAUAUGGUGCUA Cel-miR-84SEQ ID NO. 200 UGAGGUAGUAUGUAAUAUUGUASEQ ID NO. 506 UACAAUAUUACAUACUACCUCA Cel-miR-85SEQ ID NO. 201 UACAAAGUAUUUGAAAAGUCGUSEQ ID NO. 507 ACGACUUUUCAAAUACUUUGUA Cel-miR-86SEQ ID NO. 202 UAAGUGAAUGCUUUGCCACAGUSEQ ID NO. 508 ACUGUGGCAAAGCAUUCACUUA Cel-miR-87SEQ ID NO. 203 GUGAGCAAAGUUUCAGGUGUGCSEQ ID NO. 509 GCACACCUGAAACUUUGCUCAC Cel-miR-90SEQ ID NO. 204 UGAUAUGUUGUUUGAAUGCCCCSEQ ID NO. 510 GGGGCAUUCAAACAACAUAUCA Cel-miR-124SEQ ID NO. 205 UAAGGCACGCGGUGAAUGCCACSEQ ID NO. 511 GUGGCAUUCACCGCGUGCCUUA Cel-miR-228SEQ ID NO. 206 AAUGGCACUGCAUGAAUUCACGSEQ ID NO. 512 CGUGAAUUCAUGCAGUGCCAUU Cel-miR-229SEQ ID NO. 207 AAUGACACUGGUUAUCUUUUCCSEQ ID NO. 513 GGAAAAGAUAACCAGUGUCAUU Cel-miR-230SEQ ID NO. 208 GUAUUAGUUGUGCGACCAGGAGSEQ ID NO. 514 CUCCUGGUCGCACAACUAAUAC Cel-miR-231SEQ ID NO. 209 UAAGCUCGUGAUCAACAGGCAGSEQ ID NO. 515 CUGCCUGUUGAUCACGAGCUUA Cel-miR-232SEQ ID NO. 210 UAAAUGCAUCUUAACUGCGGUGSEQ ID NO. 516 CACCGCAGUUAAGAUGCAUUUA Cel-miR-233SEQ ID NO. 211 UUGAGCAAUGCGCAUGUGCGGGSEQ ID NO. 517 CCCGCACAUGCGCAUUGCUCAA Cel-miR-234SEQ ID NO. 212 UUAUUGCUCGAGAAUACCCUUUSEQ ID NO. 518 AAAGGGUAUUCUCGAGCAAUAA Cel-miR-235SEQ ID NO. 213 UAUUGCACUCUCCCCGGCCUGASEQ ID NO. 519 UCAGGCCGGGGAGAGUGCAAUA Cel-miR-236SEQ ID NO. 214 UAAUACUGUCAGGUAAUGACGCSEQ ID NO. 520 GCGUCAUUACCUGACAGUAUUA Cel-miR-237SEQ ID NO. 215 UCCCUGAGAAUUCUCGAACAGCSEQ ID NO. 521 GCUGUUCGAGAAUUCUCAGGGA Cel-miR-238SEQ ID NO. 216 UUUGUACUCCGAUGCCAUUCAGSEQ ID NO. 522 CUGAAUGGCAUCGGAGUACAAA Cel-miR-239aSEQ ID NO. 217 UUUGUACUACACAUAGGUACUGSEQ ID NO. 523 CAGUACCUAUGUGUAGUACAAA Cel-miR-239bSEQ ID NO. 218 UUUGUACUACACAAAAGUACUGSEQ ID NO. 524 CAGUACUUUUGUGUAGUACAAA Cel-miR-240SEQ ID NO. 219 UACUGGCCCCCAAAUCUUCGCUSEQ ID NO. 525 AGCGAAGAUUUGGGGGCCAGUA Cel-miR-241SEQ ID NO. 220 UGAGGUAGGUGCGAGAAAUGACSEQ ID NO. 526 GUCAUUUCUCGCACCUACCUCA Cel-miR-242SEQ ID NO. 221 UUGCGUAGGCCUUUGCUUCGAGSEQ ID NO. 527 CUCGAAGCAAAGGCCUACGCAA Cel-miR-243SEQ ID NO. 222 CGGUACGAUCGCGGCGGGAUAUSEQ ID NO. 528 AUAUCCCGCCGCGAUCGUACCG Cel-miR-244SEQ ID NO. 223 UCUUUGGUUGUACAAAGUGGUASEQ ID NO. 529 UACCACUUUGUACAACCAAAGA Cel-miR-245SEQ ID NO. 224 AUUGGUCCCCUCCAAGUAGCUCSEQ ID NO. 530 GAGCUACUUGGAGGGGACCAAU Cel-miR-246SEQ ID NO. 225 UUACAUGUUUCGGGUAGGAGCUSEQ ID NO. 531 AGCUCCUACCCGAAACAUGUAA Cel-miR-247SEQ ID NO. 226 UGACUAGAGCCUAUUCUCUUCUSEQ ID NO. 532 AGAAGAGAAUAGGCUCUAGUCA Cel-miR-248SEQ ID NO. 227 UACACGUGCACGGAUAACGCUCSEQ ID NO. 533 GAGCGUUAUCCGUGCACGUGUA Cel-miR-249SEQ ID NO. 228 UCACAGGACUUUUGAGCGUUGCSEQ ID NO. 534 GCAACGCUCAAAAGUCCUGUGA Cel-miR-250SEQ ID NO. 229 UCACAGUCAACUGUUGGCAUGGSEQ ID NO. 535 CCAUGCCAACAGUUGACUGUGA Cel-miR-251SEQ ID NO. 230 UUAAGUAGUGGUGCCGCUCUUASEQ ID NO. 536 UAAGAGCGGCACCACUACUUAA Cel-miR-252SEQ ID NO. 231 UAAGUAGUAGUGCCGCAGGUAASEQ ID NO. 537 UUACCUGCGGCACUACUACUUA Cel-miR-253SEQ ID NO. 232 CACACCUCACUAACACUGACCASEQ ID NO. 538 UGGUCAGUGUUAGUGAGGUGUG Cel-miR-254SEQ ID NO. 233 UGCAAAUCUUUCGCGACUGUAGSEQ ID NO. 539 CUACAGUCGCGAAAGAUUUGCA Cel-miR-256SEQ ID NO. 234 UGGAAUGCAUAGAAGACUGUACSEQ ID NO. 540 GUACAGUCUUCUAUGCAUUCCA Cel-miR-257SEQ ID NO. 235 GAGUAUCAGGAGUACCCAGUGASEQ ID NO. 541 UCACUGGGUACUCCUGAUACUC Cel-miR-258SEQ ID NO. 236 GGUUUUGAGAGGAAUCCUUUUASEQ ID NO. 542 UAAAAGGAUUCCUCUCAAAACC Cel-miR-259SEQ ID NO. 237 AGUAAAUCUCAUCCUAAUCUGGSEQ ID NO. 543 CCAGAUUAGGAUGAGAUUUACU Cel-miR-260SEQ ID NO. 238 GUGAUGUCGAACUCUUGUAGGASEQ ID NO. 544 UCCUACAAGAGUUCGACAUCAC Cel-miR-261SEQ ID NO. 239 UAGCUUUUUAGUUUUCACGGUGSEQ ID NO. 545 CACCGUGAAAACUAAAAAGCUA Cel-miR-262SEQ ID NO. 240 GUUUCUCGAUGUUUUCUGAUACSEQ ID NO. 546 GUAUCAGAAAACAUCGAGAAAC Cel-miR-264SEQ ID NO. 241 GGCGGGUGGUUGUUGUUAUGGGSEQ ID NO. 547 CCCAUAACAACAACCACCCGCC Cel-miR-265SEQ ID NO. 242 UGAGGGAGGAAGGGUGGUAUUUSEQ ID NO. 548 AAAUACCACCCUUCCUCCCUCA Cel-miR-266SEQ ID NO. 243 AGGCAAGACUUUGGCAAAGCUUSEQ ID NO. 549 AAGCUUUGCCAAAGUCUUGCCU Cel-miR-267SEQ ID NO. 244 CCCGUGAAGUGUCUGCUGCAAUSEQ ID NO. 550 AUUGCAGCAGACACUUCACGGG Cel-miR-268SEQ ID NO. 245 GGCAAGAAUUAGAAGCAGUUUGSEQ ID NO. 551 CAAACUGCUUCUAAUUCUUGCC Cel-miR-269SEQ ID NO. 246 GGCAAGACUCUGGCAAAACUUGSEQ ID NO. 552 CAAGUUUUGCCAGAGUCUUGCC Cel-miR-270SEQ ID NO. 247 GGCAUGAUGUAGCAGUGGAGAUSEQ ID NO. 553 AUCUCCACUGCUACAUCAUGCC Cel-miR-271SEQ ID NO. 248 UCGCCGGGUGGGAAAGCAUUCGSEQ ID NO. 554 CGAAUGCUUUCCCACCCGGCGA Cel-miR-272SEQ ID NO. 249 UGUAGGCAUGGGUGUUUGGAAGSEQ ID NO. 555 CUUCCAAACACCCAUGCCUACA Cel-miR-273SEQ ID NO. 250 UGCCCGUACUGUGUCGGCUGCUSEQ ID NO. 556 AGCAGCCGACACAGUACGGGCA

TABLE 4 Drosophila microRNA and anti-microRNA sequences. microRNA namemicroRNA sequence (5′ to 3′) Anti-microRNA molecule sequence (5′ to 3′)Dme-miR-263a SEQ ID NO. 251 GUUAAUGGCACUGGAAGAAUUCSEQ ID NO. 557 GAAUUCUUCCAGUGCCAUUAAC Dme-miR-184SEQ ID NO. 252 UGGACGGAGAACUGAUAAGGGCSEQ ID NO. 558 GCCCUUAUCAGUUCUCCGUCCA Dme-miR-274SEQ ID NO. 253 UUUUGUGACCGACACUAACGGGSEQ ID NO. 559 CCCGUUAGUGUCGGUCACAAAA Dme-miR-275SEQ ID NO. 254 UCAGGUACCUGAAGUAGCGCGCSEQ ID NO. 560 GCGCGCUACUUCAGGUACCUGA Dme-miR-92aSEQ ID NO. 255 CAUUGCACUUGUCCCGGCCUAUSEQ ID NO. 561 AUAGGCCGGGACAAGUGCAAUG Dme-miR-219SEQ ID NO. 256 UGAUUGUCCAAACGCAAUUCUUSEQ ID NO. 562 AAGAAUUGCGUUUGGACAAUCA Dme-miR-276aSEQ ID NO. 257 UAGGAACUUCAUACCGUGCUCUSEQ ID NO. 563 AGAGCACGGUAUGAAGUUCCUA Dme-miR-277SEQ ID NO. 258 UAAAUGCACUAUCUGGUACGACSEQ ID NO. 564 GUCGUACCAGAUAGUGCAUUUA Dme-miR-278SEQ ID NO. 259 UCGGUGGGACUUUCGUCCGUUUSEQ ID NO. 565 AAACGGACGAAAGUCCCACCGA Dme-miR-133SEQ ID NO. 260 UUGGUCCCCUUCAACCAGCUGUSEQ ID NO. 566 ACAGCUGGUUGAAGGGGACCAA Dme-miR-279SEQ ID NO. 261 UGACUAGAUCCACACUCAUUAASEQ ID NO. 567 UUAAUGAGUGUGGAUCUAGUCA Dme-miR-33SEQ ID NO. 262 AGGUGCAUUGUAGUCGCAUUGUSEQ ID NO. 568 ACAAUGCGACUACAAUGCACCU Dme-miR-280SEQ ID NO. 263 UGUAUUUACGUUGCAUAUGAAASEQ ID NO. 569 UUUCAUAUGCAACGUAAAUACA Dme-miR-281SEQ ID NO. 264 UGUCAUGGAAUUGCUCUCUUUGSEQ ID NO. 570 CAAAGAGAGCAAUUCCAUGACA Dme-miR-282SEQ ID NO. 265 AAUCUAGCCUCUACUAGGCUUUSEQ ID NO. 571 AAAGCCUAGUAGAGGCUAGAUU Dme-miR-283SEQ ID NO. 266 UAAAUAUCAGCUGGUAAUUCUGSEQ ID NO. 572 CAGAAUUACCAGCUGAUAUUUA Dme-miR-284SEQ ID NO. 267 UGAAGUCAGCAACUUGAUUCCASEQ ID NO. 573 UGGAAUCAAGUUGCUGACUUCA Dme-miR-34SEQ ID NO. 268 UGGCAGUGUGGUUAGCUGGUUGSEQ ID NO. 574 CAACCAGCUAACCACACUGCCA Dme-miR-124SEQ ID NO. 269 UAAGGCACGCGGUGAAUGCCAASEQ ID NO. 575 UUGGCAUUCACCGCGUGCCUUA Dme-miR-79SEQ ID NO. 270 UAAAGCUAGAUUACCAAAGCAUSEQ ID NO. 576 AUGCUUUGGUAAUCUAGCUUUA Dme-miR-276bSEQ ID NO. 271 UAGGAACUUAAUACCGUGCUCUSEQ ID NO. 577 AGAGCACGGUAUUAAGUUCCUA Dme-miR-210SEQ ID NO. 272 UUGUGCGUGUGACAGCGGCUAUSEQ ID NO. 578 AUAGCCGCUGUCACACGCACAA Dme-miR-285SEQ ID NO. 273 UAGCACCAUUCGAAAUCAGUGCSEQ ID NO. 579 GCACUGAUUUCGAAUGGUGCUA Dme-miR-100SEQ ID NO. 274 AACCCGUAAAUCCGAACUUGUGSEQ ID NO. 580 CACAAGUUCGGAUUUACGGGUU Dme-miR-92bSEQ ID NO. 275 AAUUGCACUAGUCCCGGCCUGCSEQ ID NO. 581 GCAGGCCGGGACUAGUGCAAUU Dme-miR-286SEQ ID NO. 276 UGACUAGACCGAACACUCGUGCSEQ ID NO. 582 GCACGAGUGUUCGGUCUAGUCA Dme-miR-287SEQ ID NO. 277 UGUGUUGAAAAUCGUUUGCACGSEQ ID NO. 583 CGUGCAAACGAUUUUCAACACA Dme-miR-87SEQ ID NO. 278 UUGAGCAAAAUUUCAGGUGUGUSEQ ID NO. 584 ACACACCUGAAAUUUUGCUCAA Dme-miR-263bSEQ ID NO. 279 CUUGGCACUGGGAGAAUUCACASEQ ID NO. 585 UGUGAAUUCUCCCAGUGCCAAG Dme-miR-288SEQ ID NO. 280 UUUCAUGUCGAUUUCAUUUCAUSEQ ID NO. 586 AUGAAAUGAAAUCGACAUGAAA Dme-miR-289SEQ ID NO. 281 UAAAUAUUUAAGUGGAGCCUGCSEQ ID NO. 587 GCAGGCUCCACUUAAAUAUUUA Dme-bantamSEQ ID NO. 282 UGAGAUCAUUUUGAAAGCUGAUSEQ ID NO. 588 AUCAGCUUUCAAAAUGAUCUCA Dme-miR-303SEQ ID NO. 283 UUUAGGUUUCACAGGAAACUGGSEQ ID NO. 589 CCAGUUUCCUGUGAAACCUAAA Dme-miR-31bSEQ ID NO. 284 UGGCAAGAUGUCGGAAUAGCUGSEQ ID NO. 590 CAGCUAUUCCGACAUCUUGCCA Dme-miR-304SEQ ID NO. 285 UAAUCUCAAUUUGUAAAUGUGASEQ ID NO. 591 UCACAUUUACAAAUUGAGAUUA Dme-miR-305SEQ ID NO. 286 AUUGUACUUCAUCAGGUGCUCUSEQ ID NO. 592 AGAGCACCUGAUGAAGUACAAU Dme-miR-9cSEQ ID NO. 287 UCUUUGGUAUUCUAGCUGUAGASEQ ID NO. 593 UCUACAGCUAGAAUACCAAAGA Dme-miR-306SEQ ID NO. 288 UCAGGUACUUAGUGACUCUCAASEQ ID NO. 594 UUGAGAGUCACUAAGUACCUGA Dme-miR-9bSEQ ID NO. 289 UCUUUGGUGAUUUUAGCUGUAUSEQ ID NO. 595 AUACAGCUAAAAUCACCAAAGA Dme-miR-125SEQ ID NO. 290 UCCCUGAGACCCUAACUUGUGASEQ ID NO. 596 UCACAAGUUAGGGUCUCAGGGA Dme-miR-307SEQ ID NO. 291 UCACAACCUCCUUGAGUGAGCGSEQ ID NO. 597 CGCUCACUCAAGGAGGUUGUGA Dme-miR-308SEQ ID NO. 292 AAUCACAGGAUUAUACUGUGAGSEQ ID NO. 598 CUCACAGUAUAAUCCUGUGAUU dme-miR-31aSEQ ID NO. 293 UGGCAAGAUGUCGGCAUAGCUGSEQ ID NO. 599 CAGCUAUGCCGACAUCUUGCCA dme-miR-309SEQ ID NO. 294 GCACUGGGUAAAGUUUGUCCUASEQ ID NO. 600 UAGGACAAACUUUACCCAGUGC dme-miR-310SEQ ID NO. 295 UAUUGCACACUUCCCGGCCUUUSEQ ID NO. 601 AAAGGCCGGGAAGUGUGCAAUA dme-miR-311SEQ ID NO. 296 UAUUGCACAUUCACCGGCCUGASEQ ID NO. 602 UCAGGCCGGUGAAUGUGCAAUA dme-miR-312SEQ ID NO. 297 UAUUGCACUUGAGACGGCCUGASEQ ID NO. 603 UCAGGCCGUCUCAAGUGCAAUA dme-miR-313SEQ ID NO. 298 UAUUGCACUUUUCACAGCCCGASEQ ID NO. 604 UCGGGCUGUGAAAAGUGCAAUA dme-miR-314SEQ ID NO. 299 UAUUCGAGCCAAUAAGUUCGGSEQ ID NO. 605 CCGAACUUAUUGGCUCGAAUA dme-miR-315SEQ ID NO. 300 UUUUGAUUGUUGCUCAGAAAGCSEQ ID NO. 606 GCUUUCUGAGCAACAAUCAAAA dme-miR-316SEQ ID NO. 301 UGUCUUUUUCCGCUUACUGGCGSEQ ID NO. 607 CGCCAGUAAGCGGAAAAAGACA dme-miR-317SEQ ID NO. 302 UGAACACAGCUGGUGGUAUCCASEQ ID NO. 608 UGGAUACCACCAGCUGUGUUCA dme-miR-318SEQ ID NO. 303 UCACUGGGCUUUGUUUAUCUCASEQ ID NO. 609 UGAGAUAAACAAAGCCCAGUGA dme-miR-2cSEQ ID NO. 304 UAUCACAGCCAGCUUUGAUGGGSEQ ID NO. 610 CCCAUCAAAGCUGGCUGUGAUA Dme-miR-iab45pSEQ ID NO. 305 ACGUAUACUGAAUGUAUCCUGASEQ ID NO. 611 UCAGGAUACAUUCAGUAUACGU Dme-miR-iab43pSEQ ID NO. 306 CGGUAUACCUUCAGUAUACGUASEQ ID NO. 612 UACGUAUACUGAAGGUAUACCG

EXAMPLES Example 1 Materials and Methods

Oligonucleotide Synthesis

MiR-21 were synthesized using 5′-silyl, 2′-ACE phosphoramidites(Dharmacon, Lafayette, Colo., USA) on 0.2 μmol synthesis columns using amodified ABI 394 synthesizer (Foster City, Calif., USA) (Scaringe,Methods Enzymol. 317, 3-18 (2001) and Scaringe, Methods 23, 206-217(2001)). The phosphate methyl group was removed by flushing the columnwith 2 ml of 0.2 M 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydratein DMF/water (98:2 v/v) for 30 min at room temperature. The reagent wasremoved and the column rinsed with 10 ml water followed by 10 mlacetonitrile. The oligonucleotide was cleaved and eluted from the solidsupport by flushing with 1.6 ml of 40% aqueous methylamine over 2 min,collected in a screwcap vial and incubated for 10 min at 55° C.Subsequently, the base-treated oligonucleotide was dried down in anEppendorf concentrator to remove methylamine and water. The residue wasdissolved in sterile 2′-deprotection buffer (400 μl of 100 mMacetate-TEMED, pH 3.8, for a 0.2 μmol scale synthesis) and incubated for30 minutes at 60° C. to remove the 2′ ACE group. The oligoribonucleotidewas precipitated from the acetate-TEMED solution by adding 24 μl 5 MNaCl and 1.2 ml of absolute ethanol.

2′-O-Methyl oligoribonucleotides were synthesized using 5′-DMT,2′-O-methyl phosphoramidites (Proligo, Hamburg, Germany) on 1 μmolsynthesis columns loaded with 3′-aminomodifier (TFA) C7 Icaa controlpore glass support (Chemgenes, Mass., USA). The aminolinker was added inorder to also use the oligonucleotides for conjugation to amino groupreactive reagents, such as biotin succinimidyl esters. The synthesisproducts were deprotected for 16 h at 55° C. in 30% aqueous ammonia andthen precipitated by the addition of 12 ml absolute 1-butanol. Thefull-length product was then gel-purified using a denaturing 20%polyacrylamide gel. 2′-Deoxyoligonucleotides were prepared using 0.2μmmol scale synthesis and standard DNA synthesis reagents (Proligo,Hamburg, Germany).

The sequences of the 2′-O-methyl oligoribonucleotides were5′-GUCAACAUCAGUCUGAUAAGCUAL (L, 3′ aminolinker) for 2′-OMe miR-21 (SEQID NO. 613), and 5′-AAGGCAAGCUGACCCUGAAGUL for EGFP 2′-OMe antisense(SEQ ID NO. 614), 5′-UGAAGUCCCAGUCGAACGGAAL for EGFP 2′-OMe reverse (SEQID NO. 615); the sequence of chimeric 2′-OMe/DNA oligonucleotides was5′-GTCAACATCAGTCTGATAAGCTAGCGL for 2′-deoxy miR-21 (underlined, 2′-OMeresidues) (SEQ ID NO. 616), and 5′-AAGGCAAGCTGACCCTGAAGTGCGL for EGFP2′-deoxy antisense (SEQ ID NO. 617).

The miR-21 cleavage substrate was prepared by PCR-based extension of thepartially complementary synthetic DNA oligonucleotides5′-GAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGTCAACATCAGTCTGATAAGCTATCGGTTGGCAGAAGCTAT (SEQ ID NO. 618) and5′-GGCATAAAGAATTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTCAGCCCATATCGTTTCATAGCTTCTGCCAACCGA (SEQ ID NO. 619). The extended dsDNAwas then used as template for a new PCR with primers5′-TAATACGACTCACTATAGAACAATTGCTTTTACAG (SEQ ID NO. 620) and5′-ATTTAGGTGACACTATAGGCATAAAGAATTGAAGA (SEQ ID NO. 621) to introduce theT7 and SP6 promoter sequences for in vitro transcription. The PCRproduct was ligated into pCR2.1-TOPO (Invitrogen). Plasmids isolatedfrom sequence-verified clones were used as templates for PCR to producesufficient template for run-off in vitro transcription reactions usingphage RNA polymerases (Elbashir et al., EMBO 20, 6877-6888 (2001)).³²P-Cap-labelling was performed as reported (Martinez et al., Cell 110,563-574 (2002)).

Plasmids

Plasmids pEGFP-S-21 and pEGFP-A-21 were generated by T4 DNA ligation ofpreannealed oligodeoxynucleotides 5′-GGCCTCAACATCAGTCTGATAAGCTAGGTACCT(SEQ ID NO. 622) and 5′-GGCCAGGTACCTAGCTTATCAGACTGATGTTGA (SEQ ID NO.623) into NotI digested pEGFP-N-1 (Clontech). The plasmid pHcRed-C1 wasfrom Clontech.

HeLa Extracts and miR-21 Quantification

HeLa cell extracts were prepared as described (Dignam et al., NucleicAcid Res. 11 1475-1489 (1983)). 5×10⁹ cells from HeLa suspensioncultures were collected by centrifugation and washed with PBS (pH7.4).The cell pellet (approx. 15 ml) was re-suspended in two times of itsvolume with 10 mM KC1/1.5 mM MgCl₂/0.5 mM dithiothreitol/10 mM HEPES-KOH(pH 7.9) and homogenized by douncing. The nuclei were then removed bycentrifugation of the cell lysate at 1000 g for 10 min. The supernatantwas spun in an ultracentrifuge for 1 h at 10,5000 g to obtain thecytoplasmic S100 extract. The concentration of KCl of the S100 extractwas subsequently raised to 100 mM by the addition of 1 M KCl. Theextract was then supplemented with 10% glycerol and frozen in liquidnitrogen.

280 μg of total RNA was isolated from 1 ml of 5100 extract using theacidic guanidinium thiocyanate-phenol-chloroform extraction method(Chomczynski et al., Anal. Biochem. 162, 156-159 (1987)). A calibrationcurve for miR-21 Northern signals was produced by loading increasingamounts (10 to 30000 pg) of synthetically made miR-21 (Lim et al. etal., Genes & Devel. 17, 991-1008 (2003)). Northern blot analysis wasperformed as described using 30 μg of total RNA per well (Lagos-Quintanaet al., Science 294, 853-858 (2001)).

In Vitro miRNA Cleavage and Inhibition Assay

2′-O-Methyl oligoribonucleotides or 2′-deoxyoligonucleotides werepre-incubated with HeLa S100 at 30° C. for 20 min prior to the additionof the cap-labeled miR-21 target RNA. The concentration of the reactioncomponents were 5 nM target RNA, 1 mM ATP, 0.2 mM GTP, 10 U/ml RNasin(Promega) and 50% HeLa 5100 extract in a final reaction volume of 25 μl.The reaction time was 1.5 h at 30° C. The reaction was stopped byaddition of 200 μl of 300 mM NaCl/25 mM EDTA/20% w/v SDS/200 mM Tris HCl(pH7.5). Subsequently, proteinase K was added to a final concentrationof 0.6 mg/ml and the sample was incubated for 15 min at 65° C. Afterphenol/chloroform extraction, the RNA was ethanol-precipitated andseparated on a 6% denaturing polyacrylamide gel. Radioactivity wasdetected by phosphorimaging.

Cell Culture and Transfection

HeLa S3 and HeLa S3/GFP were grown in 5% CO2 at 37° C. in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum(FBS), 100 unit/ml penicillin, and 100 μg/ml streptomycin. One daybefore transfection, 105 cells were plated in 500 μl DMEM containing 10%FBS per well of a 24-well plate. Plasmid and plasmid/oligonucleotidetransfection was carried out with Lipofectamine-2000 (Invitrogen). 0.2pEGFP or its derivatives were cotransfected with 0.3 μg pHcRed with orwithout 10 μmol of 2′-O-methyl oligoribonucleotide or 10 μmol of2′-deoxyoligonucleotide per well. Fluorescent cell images were recordedon a Zeiss Axiovert 200 inverted fluorescence microscope(Plan-Apochromat 10×/0.45) equipped with Chroma Technology Corp. filtersets 41001 (EGFP) and 41002c (HcRed) and AxioVision 3.1 software.

Example 2 MicroRNA-21 Cleavage of Target RNA

In order to assess the ability of modified oligonucleotides tospecifically interfere with miRNA function, we used our previouslydescribed mammalian biochemical system developed for assaying RISCactivity (Martinez et al., Cell 100, 563-574 (2002)). Zamore andcolleagues (Hutvàgner et al., Science 297, 2056-2050 (2002)) showed thatcrude cytoplasmic cell lysates and eIF2C2 immunoprecipitates preparedfrom these lysates contain let-7 RNPs that specifically cleavelet-7-complementary target RNAs. We previously reported that in HeLacells, numerous miRNAs are expressed including several let-7 miRNAvariants (Lagos-Quintana et al., Science 294, 853-858 (2001)).

To assess if other HeLa cell miRNAs are also engaged in RISC like miRNPswe examined the cleavage of a 32P-cap-labelled substrate RNA with acomplementary site to the highly expressed miR-21 (Lagos-Quintana etal., Science 294, 853-858 (2001); Mourelatos et al., Genes & Dev. 16,720-728 (2002)). Sequence-specific target RNA degradation was readilyobserved and appeared to be approximately 2- to 5-fold more effectivethan cleavage of a similar let-7 target RNA (FIG. 2A, lane 1, and datanot shown). We therefore decided to interfere with miR-21 guided targetRNA cleavage.

Example 3 Anti MicroRNA-21 2′-O-methyl Oligoribonucleotide InhibitedMicroRNA-21-Induced Cleavage of Target RNA

A 24-nucleotide 2′-O-methyl oligoribonucleotide that contained a 3′ C7aminolinker and was complementary to the longest form of the miR-21 wassynthesized. The aminolinker was introduced in order to enablepost-synthetic conjugation of non-nucleotidic residues such as biotin.

Increasing concentrations of anti miR-21 2′-O-methyl oligoribonucleotideand a control 2′-O-methyl oligoribonucleotide cognate to an EGFPsequence were added to the S100 extract 20 min prior to the addition of32P-cap-labelled substrate. We determined the concentration of miR-21 inthe 5100 extract by quantitative Northern blotting to be 50 μM (Lim etal., Genes & Devel. 17, 991-1008 (2003)).

The control EGFP oligonucleotide did not interfere with miR-21 cleavageeven at the highest applied concentration (FIG. 2A, lanes 2-3). Incontrast, the activity of miR-21 was completely blocked at aconcentration of only 3 nM (FIG. 2A, lane 5), and a concentration of 0.3nM showed a substantial 60%-70% reduction of cleavage activity (FIG. 2,lane 6). At a concentration of 0.03 nM, the cleavage activity of miR-21was not affected when compared to the lysate alone (FIG. 2, lane 1, 7).

Antisense 2′-deoxyoligonucleotides (approximately 90% DNA molecules) atconcentrations identical to those of 2′-O-methyl oligoribonucleotides,we could not detect blockage of miR-21 induced cleavage (FIG. 2A, lanes8-10). The 2′-deoxynucleotides used in this study were protected against3′-exonucleases by the addition of three 2′-O-methyl ribonucleotideresidues.

Example 4 Anti MicroRNA-21 2′-O-methyl Oligoribonucleotide InhibitedMicroRNA-21-Induced Cleavage of Target RNA In Vitro

In order to monitor the activity of miR-21 in HeLa cells, we constructedreporter plasmids that express EGFP mRNA that contains in its 3′ UTR a22-nt sequence complementary to miR-21 (pEGFP-S-21) or in senseorientation to miR-21 (p-EGFP-A-21). Endogenous miRNAs have previouslybeen shown to act like siRNAs by cleaving reporter mRNAs carryingsequences perfectly complementary to miRNA. To monitor transfectionefficiency and specific interference with the EGFP indicator plasmids,the far-red fluorescent protein encoding plasmid pHcRed-C1 wascotransfected.

Expression of EGFP was observed in HeLa cells transfected with pEGFP andpEGFP-A-21 (FIG. 3, rows 1 and 2), but not from those transfected withpEGFP-S-21 (FIG. 3, row 3). However, expression of EGFP from pEGFP-S-21was restored upon cotransfection with anti miR-21 2′-O-methyloligoribonucleotide (FIG. 3, row 4). Consistent with our aboveobservation, the 2′-deoxy anti miR-21 oligonucleotide showed no effect(FIG. 3, row 5). Similarly, cotransfection of the EGFP 2′-O-methyloligoribonucleotide in sense orientation with respect to the EGFP mRNA(or antisense to EGFP guide siRNA) had no effect (FIG. 3, row 6).

We have demonstrated that miRNP complexes can be effectively andsequence-specifically inhibited with 2′-O-methyl oligoribonucleotidesantisense to the guide strand positioned in the RNA silencing complex.

Incorporation of Sequence Listing

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled, “sequence_listing.txt”,created on Jun. 4, 2010. The sequence_listing.txt file is 103 kb insize.

1. An isolated molecule comprising a maximum of fifty moieties, whereineach moiety comprises a base bonded to a backbone unit, said moleculecomprising the microRNA molecule identified in SEQ ID NO: 143 or itscorresponding anti-micro RNA molecule identified in SEQ ID NO:
 449. 2. Amolecule according to claim 1, wherein the molecule is modified forincreased nuclease resistance.
 3. The molecule according to claim 1,wherein at least one of the moieties is a modified ribonucleotidemoiety.
 4. The molecule according to claim 3, wherein the modifiedribonucleotide is substituted at the 2′ position.
 5. The moleculeaccording to claim 4, wherein the substituent at the 2′ position is a C₁to C₄ alkyl group.
 6. The molecule according to claim 5, wherein thealkyl group is methyl.
 7. The molecule according to claim 5, wherein thealkyl group is allyl.
 8. The molecule according to claim 4, wherein thesubstituent at the 2′ position is a C₁ to C₄ alkoxy-C₁ to C₄ alkylgroup.
 9. The molecule according to claim 8, wherein the C₁ to C₄alkoxy-C₁ to C₄ alkyl group is methoxyethyl.
 10. The molecule accordingto claim 1, wherein at least one of the moieties is a2′-fluororibonucleotide moiety.
 11. The molecule according to claim 3,wherein the modified ribonucleotide has a methylene bridge between the2′-oxygen atom and the 4′-carbon atom.
 12. The molecule according toclaim 1, wherein the molecule comprises at least one modified moiety onthe 5′ end.
 13. The molecule according to claim 1, wherein the moleculecomprises at least two modified moieties at the 5′ end.
 14. The moleculeaccording to claim 1, wherein the molecule comprises at least onemodified moiety on the 3′ end.
 15. The molecule according to claim 1,wherein the molecule comprises at least two modified moieties at the 3′end.
 16. The molecule according to claim 1, wherein the moleculecomprises at least two modified moieties at the 5′ end and at least twomodified moieties at the 3′ end.
 17. The molecule according to claim 1,wherein the molecule comprises a nucleotide cap at the 5′ end, the 3′end or both.
 18. The molecule according to claim 1, wherein the moleculeconsists of the microRNA molecule identified in SEQ ID NO:
 143. 19. Themolecule according to claim 1, wherein the molecule consists of theanti-micro RNA molecule identified in SEQ ID NO: 449.